GADS is an adaptor protein implicated in CD3 signaling because of its ability to link SLP-76 to LAT. A GADS-deficient mouse was generated by gene targeting, and the function of GADS in T cell development and activation was examined. GADS- CD4-CD8- thymocytes exhibited a severe block in proliferation but still differentiated into mature T cells. GADS- thymocytes failed to respond to CD3 cross-linking in vivo and were impaired in positive and negative selection. Immunoprecipitation experiments revealed that the association between SLP-76 and LAT was uncoupled in GADS- thymocytes. These observations indicate that GADS is a critical adaptor for CD3 signaling.