Little is known about the molecular mechanisms that direct the transition from primitive to definitive hematopoiesis. In this study, we cocultured murine embryonic stem (ES) cells on OP9 stroma to induce hematopoietic differentiation as a model to study factors involved in the generation of adult hematopoietic stem cells (HSCs). Overexpression of the constitutively activated mutant signal transducer and activator of transcription (STAT) 5A(1*6) in ES cells facilitated the generation of cells that expressed the endothelial-hemangioblast marker Flk-1 within 5 days of coculture on OP9. The first CD41+/ CD45+/c-Kit+/Flk-1− hematopoietic cells arose in our culture conditions between days 5 and 7. Persistent activation of STAT5A greatly enhanced the generation of hematopoietic progenitors compared with controls, as determined by colony assays in methylcellulose. Moreover, whereas controls generated only a short transient wave of hematopoiesis lasting less than 3 weeks, expression of STAT5A(1*6) resulted in the generation of hematopoietic cobblestone area–forming cells (CAFCs) on OP9 that could be serially passaged onto new OP9, giving rise to second and third CAFCs that generated hematopoietic progenitors for ≥5 weeks, indicating a role for STAT5A in HSC self-renewal in vitro. Several definitive hematopoietic genes were upregulated by STAT5A (1*6), as well as Runx1/AML1, vascular endothelial growth factor, oncostatin M receptor, HoxB4, Wnt5A, Delta-like-1, and Bmi-1. Furthermore, ES-derived hematopoietic cells expressing STAT5A(1*6) contributed to myeloid-lymphoid hematopoiesis in primary and secondary nonobese diabetic–severe combined immunodeficiency recipients, although no donor-derived cells could be detected after 7 weeks in the secondary recipients. These data indicate that a persistent activation of STAT5A allows the generation of ES-derived HSCs that can, at least for an intermediate period, contribute to hematopoiesis in vivo.