Stable oncoretroviral gene transfer into hematopoietic stem cells (HSCs) provides permanent genetic disease correction. It is crucial to transplant enough transduced HSCs to compete with and replace the defective host hemopoiesis. To increase the number of transduced cells, the role of ex vivo expansion was investigated. For a possible clinical application, all experiments were carried out in serum-free media. A low-affinity nerve growth factor receptor (LNGFR) pseudotyped murine retroviral vector was used to transduce cord blood CD34(+) cells, which were then expanded ex vivo. These cells engrafted up to three generations of serially transplanted nonobese diabetic/severe combined immunodeficiency mice: 54.26% +/- 5.59%, 19.05% +/- 2.01%, and 6.15% +/- 5.16% CD45(+) cells from primary, secondary, and tertiary recipient bone marrow, respectively, were LNGFR(+). Repopulation in secondary and tertiary recipients indicates stability of transgene expression and long-term self-renewal potential of transduced HSCs, suggesting that retroviral gene transfer into HSCs, followed by ex vivo expansion, could facilitate long-term engraftment of genetically modified HSCs.