The ability of advanced-generation lentiviral vectors to transfer the green fluorescent protein (GFP) gene into human hematopoietic stem cells (HSCs) was studied in culture conditions that allowed expansion of transplantable human HSCs. Following 96 hours' exposure to flt3/flk2 ligand (FL), thrombopoietin (TPO), stem cell factor (SCF), and interleukin-6 (IL-6) and overnight incubation with vector particles, cord blood (CB) CD34⁺ cells were further cultured for up to 4 weeks. CD34⁺ cell expansion was similar for both transduced and control cells. Transduction efficiency of nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) was assessed by transplants into NOD/SCID mice. Mice that received transplants of transduced week 1 and week 4 expanded cells showed higher levels of human engraftment than mice receiving transplants of transduced nonexpanded cells (with transplants of 1 × 10⁵ CD34⁺ cells, the percentages of CD45⁺ cells were 20.5 ± 4.5 [week 1, expanded] and 27.2 ± 8.2 [week 4, expanded] vs 11.7 ± 2.5 [nonexpanded]; n = 5). The GFP⁺/CD45⁺ cell fraction was similar in all cases (12.5% ± 2.9% and 12.2% ± 2.7% vs 12.7% ± 2.1%). Engraftment was multilineage, with GFP⁺/lineage⁺ cells. Clonality analysis performed on the bone marrow of mice receiving transduced and week 4 expanded cells suggested that more than one integrant likely contributed to the engraftment of GFP-expressing cells. Serial transplantations were performed with transduced week 4 expanded CB cells. Secondary engraftment levels were 10.7% ± 4.3% (n = 12); 19.7% ± 6.2% of human cells were GFP⁺. In tertiary transplants the percentage of CD45⁺ cells was lower (4.3% ± 1.7%; n = 10); 14.8% ± 5.9% of human cells were GFP⁺, and human engraftment was multilineage. These results show that lentiviral vectors efficiently transduce HSCs, which can undergo expansion and maintain proliferation and self-renewal ability.