Retrovirus-mediated gene transfer into hematopoietic stem cells has been shown in mice, large animals, and humans. Transduction efficiency has been high in mice but has remained low in large animals and humans. Improved transduction efficiency into hematopoietic progenitor cells of large animals and humans has been achieved in vitro by enriching for CD34+ cells, adding growth factors to the transduction culture, extending the exposure time of hematopoietic cells to retrovirus particles, and by using retrovirus vectors pseudotyped with the gibbon ape leukemia virus envelope. Whether these modifications will also result in increased transduction of pluripotent hematopoietic stem cells has yet to be demonstrated by in vivo transplantation studies. Current transduction efficiency of hematopoietic stem cells in large animals and humans appears to be sufficiently high (0.1% to 1%) for gene marking studies. Efficiency needs to be further increased before gene transfer can be used for therapeutic applications.