S CID patients have been successfully treated by administration of ex vivo gene-corrected stem cells. However, despite its proven efficacy, such treatment carries specific risks and difficulties. We hypothesized that some of these drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. Indeed, in vivo intrathymic transfer of a gene that provides a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny, allowing long-term immune reconstitution. We assessed the feasibility of this approach in a murine model of ZAP-70–deficient SCID. A T cell–specific ZAP-70–expressing lentiviral vector was injected into thymi of adult ZAP-70^–/– mice without prior conditioning. This resulted in the long-term differentiation of mature TCR-αβ⁺ thymocytes, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70–expressing T cells demonstrated a partially diversified receptor repertoire and were responsive to alloantigens in vitro and in vivo. Improved treatment efficacy was achieved in infant ZAP-70^–/– mice, in which the thymus is proportionately larger and a higher percentage of prothymocytes are in cycle. Thus, intrathymic injection of a lentiviral vector could represent a simplified and potentially safer alternative to ex vivo gene-modified hematopoietic stem cell transplantation for gene therapy of T cell immunodeficiencies.