Failure of c-Myb–rescued endothelial cells to generate B lymphocytes. (A) VE-cadherin⁺CD31⁺ cells were sorted from differentiating wild-type ES cells and c-mybTet/KO ES cells as described in Figure 2. Cells were recultured for 13 days with OP9 cells and cytokines (SCF, Flt3L, IL7) in the presence (c-Myb–) or absence (c-Myb+) of Tet (1 μg/mL). Floating cells were harvested and analyzed by flow cytometry for expression of B220, CD19, and CD43. The B220⁺CD43⁺ population was gated and further analyzed for IL7Rα expression. (B) VE-cadherin⁺CD31⁺ cells sorted from differentiating wild-type ES cells and c-mybTet/KO ES cells were recultured for 13 days with OP9 cells and cytokines in the presence of different concentrations of Tet. Floating cells were harvested and analyzed by flow cytometry for expression of B220, CD19, CD43, and EGFP. Results shown are representative of at least 3 independent experiments. Numbers indicate the percentage of cells in population.