Multilineage hematopoiesis is maintained by a pool of stem cells, which ensures the formation of all blood cells and a functional immune system. To this aim, HSCs must self-renew and regulate the relative balance between self-renewal and differentiation. Gfi1 is a zinc-finger transcription factor and onco-protein, which is differentially expressed in cells the hematopoietic and immune compartment and plays important roles in development of myeloid cells and lymphoid cells. The expression pattern and the function of Gfi1 in development of adult mouse HSCs and progenitors were investigated by using Gfi1 deficient mice (Gfi1-/-), and Gfi1:GFP knock-in mice in which the Gfi1 coding region is replaced by the GFP gene. Gfi1 expression was followed by measuring green fluorescence, and it was found that Gfi1 is expressed HSCs, in CLPs and GMPs, but not in CMPs and MEPs. Consistent with the Gfi1 expression pattern, Gfi1 deficient mice show reduced frequencies of HSCs, CMPs and CLPs and an increase in the GMP population, suggesting that Gfi1 is essential for the maintenance of homeostasis of stem cell and early progenitor populations. The alteration of the number of progenitors was confirmed by in vivo (CFU-S, radioprotection) and in vitro functional assays (CFC). A reduction of the numbers and the size of CFU-S12 and moderately compromised short-term radioprotection capacity were found in Gfi1-/- bone marrow transplanted hosts. Bone marrow transplantation experiments confirm that the hematopoietic defects in Gfi1-/- mice are cell autonomous. Furthermore, when measured the reconstitution capacity of Gfi1-/- bone marrow cells against wild-type bone marrow cells in a competitive transplantation assay, we observed a severe impairment in long-term repopulating activities. This defect is not a result of a homing defect or a differentiation defect, but is correlated with an unusually high proportion of actively cycling HSCs. A large proportion of Gfi1-/- HSCs leave G0 phase to enter the cell cycle. We present evidence that a deregulation of the Gfi1 downstream effectors and cell cycle regulators p21cip1/waf1, E2F5 and E2F6 is responsible for these defects, and suggest that Gfi1 controls self-renewal and engraftment abilities of HSCs by regulating their cell cycle exit or entry and by maintaining a constant proportion of HSCs in G0 phase.