The methods available to efficiently transduce human CD34(+) hematopoietic stem cells (HSCs) derived from mobilized peripheral blood, such that they fully retain their engraftment potential and maintain high levels of transgene expression in vivo, have been unsatisfactory. The current murine retrovirus-based gene transfer systems require dividing cells for efficient transduction, and therefore the target HSCs must be activated ex vivo by cytokines to cycle, which may limit their engrafting ability. Lentivirus-based gene transfer systems do not require cell division and, thus, may allow for efficient gene transfer to human HSCs in the absence of any ex vivo cytokine stimulation. We constructed human immunodeficiency virus (HIV)-based vectors and compared them in vitro and in vivo with MuLV-based vectors in their ability to transduce unstimulated human CD34(+) HSCs isolated from mobilized peripheral blood. Both sets of vectors contained the marker gene that expresses the enhanced green fluorescent protein (EGFP) for evaluating transduction efficiency and were pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or the amphotropic murine leukemia virus envelope (A-MULV Env). The VSV-G-pseudotyped HIV-based vectors containing an internal mouse phosphoglycerate kinase promoter (PGK) were able to transduce up to 48% of the unstimulated CD34(+) cells as measured by EGFP expression. When these cells were injected into the human fetal thymus implants of irradiated SCID-hu Thy/Liv mice, up to 18% expressed EGFP after 8 weeks in vivo. In contrast, the MULV-based vectors were effective at transducing HSCs only in the presence of cytokines. Our results demonstrate that the improved HIV-based gene transfer system can effectively transduce unstimulated human CD34(+) HSCs, which can then differentiate into thymocytes and provide long-term transgene expression in vivo.