Efficient gene transfer into T lymphocytes may allow the treatment of several genetic dysfunctions of the hematopoietic system, such as severe combined immunodeficiency, and the development of novel therapeutic strategies for diseases such as cancers and acquired diseases such as AIDS. Lentiviral vectors can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) and/or by cytokines inducing resting T cells to enter in G1b phase of the cell cycle. In T-cell-based gene therapy trials performed to date, cells have been preactivated via their cognate antigen receptor (TCR). However, TCR stimulation shifts the T cells from na�ve to memory phenotype and leads to skewing of the T-cell population. Since, especially the na�ve T cells will provide a long-lasting immune reconstitution to patients these are the cells that need to be transduced for effective gene therapy. Now it is clear that use of the survival cytokines, IL-2 or IL-7, allows an efficient lentiviral vector gene transfer and could preserve a functional T-cell repertoire while maintaining an appropriate proportion of na�ve and memory T cells. In this protocol we give details on lentiviral transduction of T cells using TCR-stimulation or rIL-7 prestimulation. In addition, we describe the use of a new generation of lentiviral vectors displaying T-cell-activating ligands at their surface for targeted T-cell gene transfer.