Analysis of engraftment and differentiation by in vitro–derived progenitor T-cell subsets in FTOC. Human UCB CD34⁺CD38^−/lo HSCs were differentiated for 13 days on OP9-DL1 cells and CD34⁺CD7⁺⁺CD5⁻ (proT1) and CD34⁺CD7⁺⁺CD5⁺ (proT2) subsets were sorted by flow cytometry as indicated in panel A, and placed into FTOC (B) or placed back onto OP9-DL1 cells (C) for 19 days. Cells were harvested and analyzed for cell surface expression of CD45, CD7, CD34, CD5, CD1a, CD8, and CD4. Total cellularity of fetal thymic lobes after reconstitution ranged from 6 to 8 × 10³ or 5 to 12 × 10³ with proT1 cells or proT2 cells, respectively. Data are representative of 3 independent experiments in which 1.5 × 10⁴ sorted proT subsets were placed either into fetal thymus lobe pairs or in wells containing OP9-DL1 cells.