RapGEF2 is one of many guanine nucleotide exchange factors (GEFs) that specifically activate Rap1. Here, we generated RapGEF2 conditional knockout mice and studied its role in embryogenesis and fetal as well as adult hematopoietic stem cell (HSC) regulation. RapGEF2 deficiency led to embryonic lethality at ~ E11.5 due to severe yolk sac vascular defects. However, a similar number of Flk1(+) cells were present in RapGEF2(+/+) and RapGEF2(-/-) yolk sacs indicating that the bipotential early progenitors were in fact generated in the absence of RapGEF2. Further analysis of yolk sacs and embryos revealed a significant reduction of CD41 expressing cells in RapGEF2(-/-) genotype, suggesting a defect in the maintenance of definitive hematopoiesis. RapGEF2(-/-) cells displayed defects in proliferation and migration, and the in vitro colony formation ability of hematopoietic progenitors was also impaired. At the molecular level, Rap1 activation was impaired in RapGEF2(-/-) cells that in turn lead to defective B-raf/ERK signaling. Scl/Gata transcription factor expression was significantly reduced, indicating that the defects observed in RapGEF2(-/-) cells could be mediated through Scl/Gata deregulation. Inducible deletion of RapGEF2 during late embryogenesis in RapGEF2(cko/cko)ER(cre) mice leads to defective fetal liver erythropoiesis. Conversely, inducible deletion in the adult bone marrow, or specific deletion in B cells, T cells, HSCs, and endothelial cells has no impact on hematopoiesis.