Over the past two decades, the ability to transfer genes into hematopoietic stem cells (HSCs) has provided new insights into the behavior of individual stem cells and offered a novel approach for the treatment of various inherited or acquired disorders. At present, gene transfer into HSCs has been achieved mainly using modified retroviruses. While retrovirus-based vectors could efficiently transduce murine HSCs, extrapolation of these methods to large mammals and human clinical trials resulted in very low numbers of gene-marked engrafted cells. In addition, in vitro progenitor assays used to optimize gene transfer procedures were found to poorly predict the outcome of stem cell gene transfer. The focus rapidly turned to the development of superior and more relevant preclinical assays in human stem cell gene transfer research. Xenogeneic transplant models and large animal transplantation system have been invaluable. The development of better assays for evaluating human gene therapy protocols and a better understanding of stem cell and vector biology has culminated over the past decade in multiple strategies to improve gene transfer efficiency into HSCs. Improved gene transfer vectors, optimization of cytokine combination, and incorporation of a recombinant fragment of fibronectin during transduction are examples of novel successful additions to the early gene transfer protocols that have contributed to the first unequivocal clinical benefits resulting from genetic manipulation of HSC.