Transduction of hematopoietic stem and progenitor cells (HSPCs) with lentiviral vectors (LVs) constitutes a new therapeutic option for the treatment of various monogenic diseases affecting the lymphohematopoietic system. The development of detailed preclinical studies of gene therapy in animal disease models constitutes an essential step in expanding the application of gene therapy in a wide variety of inherited and acquired diseases. Here we describe an efficient protocol to transduce HSPCs from wild-type and Fanconi anemia mice with either gene-marking or therapeutic LVs. In this protocol, purified lineage-, Sca-1+, c-Kit+ bone marrow cells were transduced in vitro for a short period of time under conditions that facilitated efficient transduction of HSPCs capable of engrafting in transplanted recipients.