The thymus provides a specialized environment allowing the differentiation of T lymphocytes from bone marrow-derived progenitor cells. We and others have demonstrated that gene transfer into distinct thymocyte populations can be obtained, both in vivo and ex vivo, using lentiviral vectors. Here, we describe techniques for intrathymic lentiviral transduction in mice, using a surgical approach wherein the thoracic cavity is exposed as well as a significantly less invasive strategy wherein virions are directly injected through the skin. Moreover, thymocyte differentiation from murine and human progenitors is now feasible in vitro, under conditions wherein the Notch and IL-7 signaling pathways are activated. We describe methods allowing transduction of murine and human progenitors and their subsequent differentiation into more mature thymocytes. Conditions for lentiviral gene transfer into more differentiated human thymocyte subsets are also presented. Optimization of technologies for HIV-based gene transfer into murine and human thymocyte progenitors will advance strategies aimed at modulating T-cell differentiation and function in-vivo; approaches potentially targeting patients with genetic and acquired immunodeficiencies as well as immune-sensitive tumors. Furthermore, this technology will foster the progression of basic research aimed at elucidating molecular aspects of T-cell differentiation in mice and humans.