This chapter describes the methods we use to transduce mouse and human hematopoietic stem cells (HSCs) and human embryonic stem cells (hESCs). We provide detailed protocols for producing high-titer lentiviral supernatants by transient transfection and for measuring viral titers. Methods to concentrate viral supernatants to achieve a higher titer are also described. The protocols given here have been used successfully to transduce engrafting mouse and human HSCs as well as progenitor cells. These cells maintained stable transgene expression after engraftment in mice and in vivo differentiation. Human ESCs can also be transduced with a high efficiency, and transgene is expressed stably after hematopoietic differentiation.