In vitro generation of T lymphocytes from ES cell–derived Flk1⁺ progenitors. (A) Cells were harvested from day 5 or day 6 ES/OP9 cocultures, and Flk1⁺CD45^– cells, as shown, were isolated by flow cytometric cell sorting. (B) Sorted Flk1⁺ cells were seeded into host cell–depleted fetal thymic lobes (day 14 to 16) from RAG-2–deficient CD45.1 congenic mice by hanging drop, cultured overnight, and then transferred to FTOCs. Two weeks later, donor-derived reconstitution was determined by flow cytometric analysis for CD45.2 surface expression. Negative control (no donor) lobes received only media. In 2 additional experiments CD45.2⁺ cells were undetectable in control lobes (no donor) and represented 91% and 89% in lobes that received ES cell–derived Flk1⁺ precursors. (C) Evidence of donor-derived reconstitution was determined by flow cytometry in 3 independent experiments. Analysis of CD4 and CD8 surface expression is shown for CD45.2⁺ gated cells, percentages as indicated. Individual lobes were pooled for analysis. Experiments 1 and 2 were analyzed on day 14 FTOCs, and experiment 3 was analyzed on day 16 FTOCs.