Hematopoietic cell generation from c-myb knockout endothelial cells restored by c-Myb induction. (A-B) Wild-type ES cells (WT) and c-myb^–/–ES cells carrying the Tet-regulated c-myb transgene (c-mybTet/KO) were allowed to differentiate on OP9 stromal cell layers in the presence of Tet (1 μg/mL) for 5 days. VE-cadherin⁺CD31⁺CD45^–CD41^– cells were sorted by FACS and recultured (70 000 cells/well) with OP9 cells and cytokines (SCF, IL3, Epo) in the presence (c-Myb–) or absence (c-Myb+) of Tet (1 μg/mL). After 4 days, hematopoietic cells were harvested and counted (A). Mean ± SEM from 3 independent experiments are shown (**P < .01). Cells were then analyzed by flow cytometry for expression of CD45 and CD41 (B). Numbers indicate the percentage of cells in rectangular area. (C) VE-cadherin⁺CD31⁺ cells were sorted from differentiating ES cells as described under “Materials and methods.” In some cultures, c-myb transgene expression was induced from 2 days prior to sorting (right panels; c-mybTet/KO; c-Myb+> c-Myb+). Sorted cells were cultured for 9 days with OP9 cells and cytokines (SCF, IL3, Epo) in the absence of Tet. Hematopoietic cells generated were analyzed by flow cytometry for expression of lineage markers. Results shown are representative of at least 3 independent experiments. Numbers indicate the percentage of cells in population.