The lineage relationship between short-lived effector T cells and long-lived memory cells is not fully understood. We have described T-GFP mice previously, in which naive and early activated T cells express GFP uniformly, whereas cells that have differentiated into effector cytotoxic T cells selectively lose GFP expression. Here we studied antigen-specific CD8 T cell differentiation using T-GFP mice crossed to the TCR transgenic (Tg) mice P14 (specific for the lymphocytic choriomeningitis virus glycoprotein peptide, gp33-41). After activation with antigenic peptide, P14XT-GFP CD8(+) T cells cultured in high-dose IL-2 developed into cells with effector phenotype and function: they were blastoid, lost GFP expression, expressed high levels of activation and effector markers, and were capable of immediate cytotoxic function. In contrast, cells cultured in IL-15 or low-dose IL-2 never developed into full-fledged effector cells. Rather, they resembled memory cells: they were smaller, were GFP(+), did not express effector markers, and were incapable of immediate cytotoxicity. However, they mediated rapid-recall responses in vitro. After adoptive transfer, they survived in vivo for at least 10 weeks and mounted a secondary immune response after antigen rechallenge that was as potent as endogenously generated memory cells. In addition to providing a simple means to generate memory cells in virtually unlimited numbers, our results suggest that effector differentiation is not a prerequisite for memory cell generation.