YS and P-Sp have autonomous T-cell potential. (A) E9.5 YS and P-Sp cells from WT and Ncx1^−/− embryos were plated on OP9-DL1 stromal cells with added IL-7. After 10-14 days of coculture, CD4⁺CD8⁺ DP cells as well as TCRβ and TCRγδ⁺ were produced in each culture. (B) YS and P-Sp cells at 4-sp stage also produced DP as well as CD8⁺ cells in OP9-DL1 coculture. Representative FACS dot plots are depicted for n = 5 in each YS and P-Sp grouping. (C) TCRVγ3 expression was examined by flow cytometry with (i) E15.5 fetal thymocytes, (ii) cells derived from E14.5 fetal liver lin⁻c-kit⁺Sca-1⁺ (KSL) cells in OP9-DL1 culture, (iii) cells derived from adult BM KSL cells in OP9-DL1 culture, (iv) E9.5 YS-derived cells, and (v) E9.5 P-Sp–derived cells. TCRγδ⁺ cells were gated for each dot plot analysis. (D) RT-PCR analysis detecting TCR Vγ3, Vγ4, and Vγ5 transcripts. (1) Day 10 neonatal thymocytes, (2) E15.5 thymocytes, and cultured cells in OP9-DL1 derived from (3) 4 sp YS, (4) 4 sp P-Sp, (5) E9.5 YS, (6) E9.5 P-Sp, (7) E14.5 fetal liver KSL, and (8) E15.5 fetal liver common lymphoid progenitor (CLP) cells.