High-titer, HIV-1–based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34⁺ cells and clonogenic progenitors very poorly (< 1%), reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier, we developed a simian immunodeficiency virus (SIV)–based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% ± 1% of rhesus bulk CD34⁺ cells and 75% ± 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector–mediated stem cell gene transfer in vivo, 3 rhesus macaques underwent transplantation with transduced, autologous cytokine-mobilized peripheral blood CD34⁺ cells following myeloablative conditioning. Hematopoietic reconstitution was rapid, and an average of 18% ± 8% and 15% ± 7% GFP-positive granulocytes and monocytes, respectively, were observed 4 to 6 months after transplantation, consistent with the average vector copy number of 0.19 ± 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models.