Hematopoietic stem cells (HSCs) were among the first targets of genetic manipulation for the purpose of treating human diseases. The translational aspects of the first human clinical trials were based on results obtained using the mouse as an experimental model. Murine studies have shown that the major limitations of HSC gene therapy are similar to those encountered when using non-hematopoietic cells as targets and include (1) an inability to genetically modify sufficient numbers of target cells, (2) the loss of transgene function over time, and (3) potential complications due to vector integration. With continued improvements in transduction protocols, murine HSC transduction and transplantation are now routine with transduction efficiencies >50% easily achievable and even >90% feasible. However, attaining high-level engraftment of gene-modified cells after transplantation is still problematic. Basic transduction conditions entail cytokine stimulation of HSC populations, such as stem cell antigen-1 positive (Sca-1(+)) cells isolated from bone marrow, in serum-free media followed by multiple additions of recombinant retrovirus. Analysis of peripheral blood 12 weeks post transplantation of transduced cells into lethally irradiated recipients shows genetic marking in all hematopoietic lineages. Transduction of HSCs is then confirmed by transplanting bone marrow cells harvested from primary transplant recipients into lethally irradiated secondary recipients. Analysis of these mice shows that recombinant retroviruses transduce murine HSCs efficiently and stably and that the genetically modified cells are capable of completely repopulating the hematopoietic system.