Itk Is a T cell protein tyroslne klnase (PTK) that, along with Btk and Tee, belongs to a family of cytoplasmlc PTKs wlth N-terminal pleckstrln homology domains. Btk plays a critical role In B lymphocyte development. To determine whether Itk has an analogous role In T lymphocytes, we used gene targetlng to prepare mice lacking expression of Itk. Such animals had decreased numbers of mature thymocytes, an effect most clearly observed in mice expressing T cell receptor (TCR) transgenes. Mature T cells from Itk-deficient mice had reduced proliferative responses to allogeneic MHC stlmulation and to anti-TM cross-llnking, but responded normally to stimulation with phorbol ester plus ionomycin or with IL-2. These results provide genetic evidence that Itk Is involved in T cell development and also suggest that Itk has an Important role in proxlmal events In TCR-medlated signaling pathways.