Hematopoietic stem cells (HSC) require extensive cytokine-mediated stimulation and proliferation for efficient transduction by oncoretroviral vectors. Since lentiviral vectors can transduce nondividing cells, the need for cytokine stimulation has been questioned. We studied HIV-based lentiviral transduction of human early hematopoietic progenitors from umbilical cord blood in the presence or absence of IL-3, IL-6, stem cell factor (SCF), and Flt-3L (36SF) or SCF alone and characterized the effects of these conditions on the stem cell phenotype. Gene transfer was significantly higher in the presence of 36SF in mass culture cells, CFC, LTCIC, and NOD/SCID repopulating cells (SRC). Transduction of primitive progenitor/stem cells was poor without cytokines, with only 12% LTCIC and 23% SRC transduced, compared to 59% in LTCIC and 81% in SRC with 36SF. SCF alone matched transduction rates of multiple cytokines with 70% in CFC. Cytokines prevented apoptosis, expanded CD34⁺ cell number, and maintained CFC and LTCIC frequencies. Cytokine stimulation increased transduction of nondividing Ara-C-resistant and aphidicolin-inhibited cells similar to dividing cells. These data suggest that cytokines enhance lentiviral transduction of HSC, without requiring cell division, and maintain the stem cell phenotype. SCF stimulation alone was sufficient for high level transduction.