Signal transducers and activators of transcription (STATs) are intracellular mediators of cytokine receptor signals. Because many early-acting growth factors have been implicated in STAT5 activation, this study sought to investigate whether STAT5 may be a transcriptional regulator of hematopoietic stem cell (HSC) long-term repopulating activity. To test this possibility, bone marrow (BM) and fetal liver (FL) cells from mice containing homozygous deletions of both STAT5a and STAT5b genes (STAT5ab^−/−) were characterized for hematopoietic repopulating activities. BM and FL grafts were capable of repopulating lymphoid and myeloid lineages of lethally irradiated primary and secondary hosts, with defects observed primarily in T-lymphocyte engraftment. Because only a fraction of normal HSC function is required to reconstitute hematopoiesis, competitive repopulation assays of adult BM or FL cells were used against wild type adult BM or FL cells to quantitate stem cell function. In these analyses, average 25-, 28-, 45-, and 68-fold decreases in normal repopulating activity were evident in granulocyte (Gr-1⁺), macrophage (Mac-1⁺), erythroid progenitor (Ter119⁺), and B-lymphocyte (B220⁺) populations, respectively, with T lymphocytes (CD4⁺) always undetectable from the STAT5ab^−/− graft. Consistent with previous reports of divergence between stem cell phenotype and function in cases of perturbed hematopoiesis, the absolute number of cells within Sca-1⁺c-kit⁺lin⁻ or lin⁻ Hoechst 33342 side population fractions was not significantly different between wild type and STAT5ab^−/−BM or FL cells. These results demonstrate that a significant proportion of the growth factor signals required for multilineage reconstitution potential of HSCs is STAT5 dependent.