SIRT1 is a founding member of a sirtuin family of seven proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1-/- mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1-/- mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1+/+, +/-, and -/- mice. SIRT1-/- ESCs formed fewer mature blast cell colonies. Replated SIRT1-/- BL-CFCs demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1-/- derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1-/- ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased ?-H1 globin, ?-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1-/- ESC differentiation deficiencies.