Accumulated data indicate that current generation lentiviral vectors, which generally utilize an internal human cytomegalovirus (CMV) immediate early region enhancer–promoter to transcribe the gene of interest, are not yet optimized for efficient expression in human hematopoietic stem/progenitor cells (HSPCs). As a first step toward this goal, we constructed self-inactivating derivatives of the HIV-1-based transfer vector pHR' containing the enhanced green fluorescent protein (GFP) gene as reporter and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). GFP expression was driven by a variety of strong viral and cellular promoters, including the murine stem cell virus (MSCV) long terminal repeat (LTR), a Gibbon ape leukemia virus (GALV) LTR, the human elongation factor 1α (EF1α) promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken β-actin promoter), and the human phosphoglycerate kinase 1 (PGK) promoter. In contrast to results obtained in human embryonic kidney 293T cells and fibrosarcoma HT1080 cells, in which the CMV promoter expressed GFP at the highest levels, significantly higher levels of GFP expression (3- to 5-fold) were achieved with the MSCV LTR, the EF1α promoter, and the CAG promoter in the human HSPC line KG1a. Removal of the WPRE indicated that it stimulated GFP expression from all of the vectors in KG1a cells (up to 3-fold), although it only marginally improved the performance of the intron-containing EF1α and CAG promoters (<1.5-fold stimulation). The vectors using the MSCV LTR, the GALV LTR, and the PGK promoter were the most efficient at transducing primary human CD34+ cord blood progenitors under the conditions employed. High-level GFP expression in the NOD/SCID xenograft model was demonstrated with the pHR' derivative bearing the MSCV LTR. These new lentiviral vector backbones provide a basis for the rational design of improved delivery vehicles for human HSPC gene transfer applications.