Definitive hematopoiesis has been proposed to arise from hemogenic endothelial cells during mouse embryogenesis. The c-myb proto-oncogene is essential for the development of definitive hematopoiesis and was reported to be activated in hemogenic endothelial cells. To investigate whether c-Myb is involved in regulating the development of hemogenic endothelial cells, we conditionally induced c-myb over-expression during the in vitro differentiation of embryonic stem cells. VE-cadherin+ CD45− cells inducibly expressing c-Myb showed an increase in multilineage colony formation as well as an augmented capacity of the colony forming cells to self-renew in vitro under the condition that only the endogenous c-myb gene was expressed during differentiation of hematopoietic cells. Over-expression of c-Myb in the endothelial population led to activation of genes associated with definitive hematopoiesis such as Runx1, Hoxb4, Mll and Etv6. Our data provide evidence that c-Myb is able to exert an effect in endothelial cells which fosters the establishment of their hemogenic potential.