Schematic for the differentiation of ES cells into lymphocytes. ES cells were induced to differentiate on the bone marrow–derived stromal cell line OP9. Initial seeding of ES cells onto OP9 cells is designated as day 0. After 5 to 6 days of coculture, mesoderm-like colonies were observed and were disaggregated by treatment with 0.25% trypsin. Reseeding onto fresh OP9 cell monolayers allows for the generation of B lymphocytes. For the generation of T lymphocytes, cells were seeded into fetal thymic lobes by “hanging drop” for 1 to 2 days and then transferred to fetal thymic organ cultures (FTOCs) for approximately 2 weeks. Flow cytometric analysis of B-cell lineage markers (CD19 and CD45) and T-cell lineage markers (CD4 and CD8) are shown for a day 19 ES/OP9 coculture and a day 14 FTOC, respectively. Using this protocol, the generation of B lymphocytes is routinely observed, whereas T lymphocytes are not generated.