Secreted growth factors are integral components of the bone marrow (BM) niche and can regulate survival, proliferation, and differentiation of committed hematopoietic stem cells (HSCs). However, downstream genes activated in HSCs by early-acting cytokines are not well characterized. To better define intracellular cytokine signaling in HSC function, we have analyzed mice lacking expression of both signal transducer and activator of transcription 5a (STAT5a) and STAT5b (STAT5ab−/−). These studies specifically avoided possible autoimmune and/or splenomegaly disease-mediated indirect effects on HSC function by using 2 independent approaches: (1) by crossing onto the C57Bl/6 RAG2−/− background, and (2) by generation of wild-type chimeric mice reconstituted with transplanted STAT5ab−/− BM cells. These experiments demonstrated that STAT5-deficient HSCs have cell autonomous defects in competitive long-term repopulating activity. Furthermore, in the chimeric mice, injected wild-type BM cells showed a progressive multilineage competitive repopulating advantage in vivo, demonstrating that steady-state hematopoiesis was also highly STAT5-dependent. Consistent with the in vivo repopulating deficiency, when Sca-1+c-kit+lin− (KLS) cells were isolated and stimulated with growth factors in vitro, up to a 13-fold reduced expansion of total nucleated cells was observed in response to cocktails containing interleukin 3 (IL-3), IL-6, stem cell factor (SCF), Flt3 ligand, and thrombopoietin. Notably, a 10-fold reduction in expansion was observed with IL-3 and SCF. However, STAT5 activation was not required for regeneration of the KLS pool in vivo following transplant or for secondary repopulating ability. These studies support a major role for STAT5 activation as a cellular determinant of cytokine-mediated HSC repopulating potential but not self-renewal capacity.