If present in sufficient numbers, could extrathymic T cells substitute for thymus-derived T cells? To address this issue, we studied extrathymic T cells that develop in athymic mice under the influence of oncostatin M (OM). In this model, extensive T-cell development is probably due to amplification of a minor pathway of T-cell differentiation taking place only in the lymph nodes. Extrathymic CD4 T cells expanded poorly and were deficient in providing B-cell help after infection with vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV). Compared with classic T cells, stimulated extrathymic CD8 T cells produced copious amounts of interferon γ (IFN-γ), and their expansion was precocious but of limited amplitude because of a high apoptosis rate. Consequently, although extrathymic cytotoxic T lymphocytes (CTLs) responded to LCMV infection, as evidenced by the expansion of GP33-41 tetramer-positive CD8 T cells, they were unable to eradicate the virus. Our data indicate that the site of development impinges on T-cell quality and function and that extrathymic T cells functionally cannot substitute for classical thymic T cells. (Blood. 2004;103:3102-3110)