NATURE IMMUNOLOGY VOLUME 15 NUMBER 1 JANUARY 2014 27 ARTICLES T lymphocytes, unlike other hematopoietic cells, differentiate in the thymus, a specialized organ that provides a unique environment for the acquisition of immunological competence. Analysis of the pheno- type and stage of differentiation of the thymus-settling progenitors (TSPs) is important for characterization of the thymus-independent stages of T cell development and for devising strategies for cell- replacement therapy in lymphopenic patients. Although many studies have attempted to identify TSPs, reports over time have been con- tradictory. Depending on the experimental system, TSPs have been defined as T cell– and natural killer (NK) cell–committed cells, com- mon lymphoid progenitors (CLPs) or lymphoid-primed multipotent progenitors (LMPPs). Integrating these conflicting data has proven challenging 1 . The identification of the CLP 2–5 in the bone marrow (BM) led to the proposal that TSPs are derived from a lymphoid cell–committed popu- lation. That was disputed by the observation that mice deficient in the transcription factor Ikaros, which lack CLPs in the BM, still have early thymic progenitors (ETPs), the most immature thymocytes derived from TSPs, which suggests that hematopoietic progenitor cells more undifferentiated than CLPs colonize the thymus 6 . Evidence that ETPs from adult mice generate myeloid cells, as well as B cells, in culture has further strengthened the view that ETPs are not derived from CLPs 7,8 . A fate-map analysis, however, found almost no myeloid cells derived from cells expressing CD127 (the interleukin 7 receptor α-chain) 9 , which indicates that the myeloid potential observed in ETPs does not contribute substantially to the myeloid lineage in vivo. Moreover, under particular culture conditions that include stromal cells that express Delta-like 4 (DL4), a ligand for the Notch family of signaling receptors 7 , even CLPs can generate myeloid cells 9 , which suggests that the results noted above 7,8 could be explained by the artificial induc- tion, in vitro, of nonphysiological differentiation pathways. Analysis of thymocytes obtained from mouse embryos at embry- onic days 11–12 (E11–E12), before the thymus is structurally well defined, has shown that TSPs are engaged in the T cell–NK cell path- ways, which suggests that commitment occurs before contact with the thymic epithelium 10–12 . Consistent with that view, several laboratories have characterized progenitors of T cells–NK cells in the embryo and have found that cells with similar restricted potential are present in fetal blood (FB) 13,14 and fetal liver (FL) 15–18 . However, in newborn mice, ETPs display robust potential to develop into T cells, B cells and myeloid cells but no potential to develop into megakaryocytes or erythrocytes and thus functionally resemble LMPPs 19 . Thymocyte differentiation starts with CD4 - CD8 - double-negative (DN) cells, which also lack expression of the invariant signaling pro- tein CD3. At this stage progenitor cells expand their populations, rearrange the δ-, γ- and β-chains of their T cell antigen receptors (TCRs)(the DN2–DN3 stage) and undergo pre-TCR selection (the DN3–DN4 stage). The most immature DN stage (DN1) is character- ized by expression of the activation and memory marker CD44 and no expression of the T cell–activation marker CD25. The combination of expression of the stem cell factor receptor CD117 (c-Kit) 6,20 and B cell–differentiation marker CD24 (HSA) 21,22 can be used to further subdivide the DN1 compartment in five subsets in which DN1a and DN1b correspond to ETPs, while the other subsets have low T cell potential. Under physiological conditions, persistent thymic input is required for continuous T cell production 23,24 , but analysis of TSPs in the adult thymus is difficult because of the low number of cells that reach the thymus every day (estimated to be less than ten cells) 25 . 1 Unit for Lymphopoiesis, Immunology Department, INSERM U668 Paris, France. 2 Université Pierre et Marie Curie, Paris, France. 3 Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France. 4 Present address: INSERM U845, Research Center Growth and Signaling, Faculty-Institute Cochin, Paris, France. 5 These authors contributed equally to this work. Correspondence should be addressed to A.C. (ana.cumano@pasteur.fr). Received 16 September; accepted 6 November; published online 8 December 2013; doi:10.1038/ni.2782 Two waves of distinct hematopoietic progenitor cells colonize the fetal thymus Cyrille Ramond 1,2,4 , Claire Berthault 1,3,5 , Odile Burlen-Defranoux 1,5 , Ana Pereira de Sousa 1 , Delphine Guy-Grand 1 , Paulo Vieira 1 , Pablo Pereira 1 & Ana Cumano 1 The generation of T cells depends on the migration of hematopoietic progenitor cells to the thymus throughout life. The identity of the thymus-settling progenitor cells has been a matter of considerable debate. Here we found that thymopoiesis was initiated by a first wave of T cell lineage–restricted progenitor cells with limited capacity for population expansion but accelerated differentiation into mature T cells. They gave rise to ab and gd T cells that constituted V g 3 + dendritic epithelial T cells. Thymopoiesis was subsequently maintained by less-differentiated progenitor cells that retained the potential to develop into B cells and myeloid cells. In that second wave, which started before birth, progenitor cells had high proliferative capacity but delayed differentiation capacity and no longer gave rise to embryonic gd T cells. Our work reconciles conflicting hypotheses on the nature of thymus-settling progenitor cells. npg © 2014 Nature America, Inc. All rights reserved.