T/NK progenitors fully mature on transfer to Rag2^−/−γ_c^−/− mice. mCD34⁺ were differentiated on monolayers of TSt-4 expressing hDLL for 4 weeks, after which they were purified by cell sorting and injected intrahepatically into newborn Rag2^−/−γ_c^−/− mice. (A) Both hDLL1- and hDLL4-derived T/NK progenitors are found exclusively in the thymus at both time points analyzed and have progressed toward the CD4⁺CD8⁺ DP stage at 4.5 weeks. A limited number of DP and few CD8 single-positive (SP) cells already have CD3 surface expression (supplemental Figures 3-4). The CD4⁺ cells at this time point are immature single-positive cells (ISP); sCD3⁻ and CD5⁺CD7⁺. At 6.5 weeks, the percentages of huCD45⁺ have dropped sharply, but there is clear surface expression of sCD3 and TCR-αβ on T/NK progenitor-derived cells, on both DP and CD4/CD8 SP cells (supplemental Figure 4). (B-C) Next to T/NK progenitors, mice were given mCD34⁺ or CB-CD34⁺ cells. (B) Thymus compartment was analyzed at indicated time points. Thymocyte subsetting is based on coexpression of CD5 and CD7 only (double-negative), CD4⁺sCD3⁻ (ISP), CD4⁺CD8⁺ (DP), and CD4⁺sCD3⁺ or CD8⁺sCD3⁺ (SP). The left 2 bars of each graph represent the data from mice displayed in panel A. Compared with mCD34⁺ and CB-CD34⁺ cells, predifferentiation of mCD34⁺ cells results in a higher percentage of cells at the DP stage at 4.5 weeks, but few cells are left at 6.5 weeks. (C) The total number of huCD45⁺ cells/tissue shows that T/NK progenitors are present temporarily and exclusively in the thymus.