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Lentiviral vectors transduce at high efficiency human hematopoietic stem/progenitor cells (HSCs) without compromising their self-renewing and repopulation capacity. Last-generation helper constructs and self-inactivating (SIN) vectors have minimized the chances of generating recombinant, replication-competent HIV derivatives during packaging, and drastically reduced the potential risks of insertional activation of oncogenes. To allow the clinical use of lentiviral vectors, development of packaging cell lines and standardized procedures for vector manufacturing, downstream processing and quality/safety testing appears necessary. However, generation of stable packaging cell lines has been hampered so far by the high toxicity of VSV-G and of some of the HIV helper proteins used in the packaging steps. More recently, lentiviral vectors have been pseudotyped with alternative, non-toxic envelope proteins such as derivatives of the RD114 feline leukemia virus envelope glycoprotein, allowing the generation of a prototype, stable packaging cell line (Ikeda et al., Nature Biotech 21:569, 2003). The receptor for RD114 is abundant on human hematopoietic cells, allowing efficient transduction by RD114-pseudotyped lentiviral vectors. We report a comparison between lentiviral vector preparations pseudotyped with VSV-G and the RD114-TR fusion glycoprotein in transducing human cord blood-derived CD34+ HSCs and peripheral blood lymphocytes (PBLs). RD114-TR-pseudotyped vectors transduce both cell types at much lower m.o.i than VSV-G-pseudotyped vectors (5–10 vs. 50–200), resulting in lower toxicity, higher persistence and less vector copy number per genome. In addition, RD114-TR-pseudotyped vectors transduce at higher efficiency human clonogenic progenitors (BFU-E, CFU-GM, CFU-GEMM), as assayed in semi-solid cultures in vitro. Transduction efficiency and average vector copy number in human repopulating HSCs and their progeny was determined after xeno-transplantation in NOD-SCID mice. Our results indicate that pseudotyping with RD114-TR is a promising alternative to VSV-G for developing clinical-grade lentiviral vector preparations aimed at genetic modification of human HSCs.