Hematopoietic stem cells (HSCs) are clonogenic cells capable of both selfrenewal and multilineage differentiation. An efficient method for gene transfer into HSCs is required for exploring HSC biology as well as for gene therapy of hematopoietic disorders. Retroviral vectors have been the most widely used vectors for gene transfer to HSCs. However, retroviral vectors require cell division for integration, limiting their use for gene transfer into HSCs that are exclusively quiescent. Although prestimulation of HSCs with cytokines can enhance gene-transfer efficiency, exposure to cytokines also stimulates HSCs to differentiate, resulting in the reduction of long-term repopulating capacity (8-15). In contrast, lentiviral vectors based on the human immunodeficiency virus type 1 (HIV-1) can efficiently transduce human CD34+ cells without cytokine prestimulation and long-term multilineage expression of the transgene is detected in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice after transplantation (16-22). Murine HSCs can also be easily transduced with lentiviral vectors without cytokine prestimulation (23-26). In this chapter, the protocols for transduction of human CD34+ cells that contain HSCs are described. The procedures include the isolation of human CD34+ cells, preparation of lentiviral vectors, transduction of human CD34+ cells, and in vivo analysis of transduced HSCs using NOD/SCID mice.