Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (Kit W41/W41) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult Kit W41/W41 mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin− Sca-1+ Kit high (LSK) CD34− Flt3− long-term HSCs by 12 wk of age, whereas LSKCD34+ Flt3− short-term HSCs and LSKCD34+ Flt3+ multipotent progenitors were less affected. Whereas homing and initial reconstitution of Kit W41/W41 bone marrow cells in myeloablated recipients were close to normal, self-renewing Kit W41/W41 HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation Kit W41/W41 HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.