The low frequency of transplantable hematopoietic stem cells in adult human bone marrow (BM) and other differences from cord blood stem cells have impeded studies to optimize the retroviral transduction of stem cells from adult sources. To address this problem, first a cytokine combination was defined that would both maximize the kinetics of adult BM CD34⁺CD38⁻ cell mitogenesis and minimize the period of prestimulation required for the transduction of these cells by a MSCV-GFP/neo^r virus in tissue culture dishes in the absence of fibronectin. Three days of stimulation with flt3-ligand, Steel factor, interleukin (IL)-3, and hyper-IL-6 proved both necessary and sufficient to obtain 83% ± 2% GFP⁺ CD34⁺CD38⁻ cells, 75% ± 10% G418-resistant clonogenic progenitors, and 50% ± 20% transduced long-term culture-initiating cells as recovered 48 hours after a single exposure to virus. Moreover, this was accompanied by a several-fold increase in viral receptor (pit-1) messenger RNA transcripts in the target cells. Using this prestimulation protocol, repeated daily exposure to new virus (3×) did not alter the proportion of transduced cells over that obtained with a single exposure. Adult human BM cells able to engraft immunodeficient (NOD/SCID-β₂M^−/−) mice were also efficiently transduced (10%-20% GFP⁺ human lymphoid and myeloid cells present 6-8 weeks after transplant) using a 6-day prestimulation and infection protocol. A clinically useful efficiency of retrovirus-mediated gene transfer to transplantable adult human BM stem cells can thus be obtained with a protocol that allows their semisynchronous activation into cycle and concomitant increased expression of virus receptor transcripts before virus exposure.