The T-lymphocyte pool can be subdivided into naïve (Tn), effector memory (Tem), and central memory (Tcm) T cells. In this study, we characterized in vitro short-term cultured anti-tumor human T lymphocytes generated by lentiviral transduction with an anti-tumor antigen TCR vector. Within 2 weeks of in vitro culture, the cultured T cells showed a Tcm-like phenotype illustrated by a high percentage of CD62L and CD45RO cells. When the cells were sorted into populations that were CD45RO+/CD62L-(Tem), CD45RO+/CD62L+(Tcm), or CD45RO(low)/CD62L+(Tn) and co-cultured with antigen-matched tumor lines, the magnitude of cytokine release from these populations for IFNγ (Tn < Tcm < Tem) and IL-2 (Tn > Tcm > Tem) mimicked the types of immune cell responses observed in vivo. In comparing cell-mediated effector function, Tn were found to be deficient (relative to Tcm and Tem) in the ability to form conjugates with tumor cells and subsequent lytic activity. Moreover, analysis of the gene expression profiles of the in vitro cultured and sorted T-cell populations also demonstrated patterns consistent with their in vivo counterparts. When Tcm and Tem were tested for the ability to survive in vivo, Tcm displayed significantly increased engraftment and persistence in NOD/SCID/γc(-/-) mice. In general, a large percentage of in vitro generated anti-tumor T lymphocytes mimic a Tcm-like phenotype (based on phenotype, effector function, and increased persistence in vivo), which suggests that these Tcm-like cultured T cells may be optimal for adoptive immunotherapy.