The ability to efficiently transduce hematopoietic stem cells (HSC) represents a powerful methodology by which to study the role of specific genes on HSC function, as well as to broaden the potential of gene therapy for hematopoietic related diseases. While retroviruses have been used extensively to transduce a variety of cell types, HIV-derived lentiviruses prove superior for transduction of quiescent HSC due to their ability to infect non-dividing cells. Quality of lentiviral supernatants and starting cells are vital to obtain reproducible consistent results, and therefore, here we describe the production of concentrated lentiviral preparations, the purification of HSC from total mouse bone marrow, and their transduction to obtain long-term HSC engraftment with persistent gene transfer and expression of the desired transgene.