CD3-targeted lentiviral vectors (CD3-LVs) mediate selective transduction of human T lymphocytes in vitro and in vivo while simultaneously activating the targeted cells. Previously, we have demonstrated that CD3-LV leads to downmodulation of the CD3:T cell receptor (TCR) complex. We therefore hypothesized that inhibition of CD3 phosphorylation by Src/Abl tyrosine kinase inhibitors such as dasatinib results in enhancement of gene delivery by T cell-targeted LVs. Indeed, dasatinib treatment of T cells prior to incubation with CD3-LV increased reporter gene delivery by 3- to 10-fold. Moreover, the presence of dasatinib enhanced selective transduction into non-activated target cells present in whole blood. When combined with delivery of the CD19-chimeric antigen receptor (CAR) gene, dasatinib increased CAR T cell numbers by close to 10-fold. Importantly, the short-term exposure of T cells to dasatinib during vector incubation did not interfere with tumor cell killing by the resulting CAR T cells and rather came along with less upregulated exhaustion markers and a more naive phenotype. Our data suggest that dasatinib prevents CD3-LV-induced phosphorylation and CD3:TCR intake, thereby increasing the amount of CD3-LV bound to the cell surface. This is the first description of dasatinib as transduction enhancer, an activity particularly relevant for CAR T cell generation with CD3-LV. Display omitted This paper describes the discovery of dasatinib as transduction enhancer for gene delivery into T lymphocytes by CD3-targeted lentiviral vectors. Dasatinib acts through preventing CD3:TCR intake, thereby increasing the amount of vector particles bound to the T cell surface. Improved generation of CAR T cells is illustrated as an immediate application.