The hematopoietic stem/progenitor cell (HSPC) represents the ideal target for gene therapy of disorders of the hematopoietic system, but still faces problems related to ex vivo manipulation and gene transfer efficiency. We demonstrate that soluble factors from the human endothelial-like cell line ECV 304/T24 support the growth of human CD34(+) progenitor cells as primary human bone marrow stroma and increase the rate of gene transfer into progenitor cells up to 5-fold. ECV 304/T24 was used to generate split-function amphotropic packaging cell lines (named APEX) with the purpose of combining, in the same cells, hematopoietic support and gene transfer vehicle functions. The APEX cell lines were negative for the presence of replication-competent retroviruses and produced complement-resistant vector particles. When mobilized peripheral blood or umbilical cord blood CD34(+) cells were exposed once to APEX supernatants, the level of gene transfer was equivalent to that observed with GP + Am12, in spite of the lower titer of the APEX producers. More importantly, APEX supernatants gave rise reproducibly to a 2-fold increase in transduction of early progenitors (long-term culture-initiating cells), reaching on average 50% gene transfer. This novel packaging cell represents a significant advance in HSPC genetic modification technology, combining both a beneficial hematopoietic supportive effect and the gene transfer vector function in a human-based system.