The transcription factor STAT5 fulfills a distinct role in the hematopoietic system, but its precise role in primitive human hematopoietic cells remains to be elucidated. Therefore, we performed STAT5 RNAi in sorted cord blood (CB) and acute myeloid leukemia (AML) CD34⁺ cells by lentiviral transduction and investigated effects of STAT5 downmodulation on the normal stem/progenitor cell compartment and the leukemic counterpart. STAT5 RNAi cells displayed growth impairment, without affecting their differentiation in CB and AML cultures on MS5 stroma. In CB, limiting-dilution assays demonstrated a 3.9-fold reduction in progenitor numbers. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays, and the average LTC-IC frequency was 3.25-fold reduced from 0.13% to 0.04% by STAT5 down-regulation. Single-cell sorting experiments of CB CD34⁺/CD38⁻ cells demonstrated a 2-fold reduced cytokine-driven expansion, with a subsequent 2.3-fold reduction of progenitors. In sorted CD34⁺ AML cells with constitutive STAT5 phosphorylation (5/8), STAT5 RNAi demonstrated a reduction in cell number (72% ± 17%) and a decreased expansion (17 ± 15 vs 80 ± 58 in control cultures) at week 6 on MS5 stroma. Together, our data indicate that STAT5 expression is required for the maintenance and expansion of primitive hematopoietic stem and progenitor cells, both in normal as well as leukemic hematopoiesis.