Despite of the potential implications for cancer immunotherapy, conventional approaches using in vitro expanded CD8(+) T cells have suboptimal outcomes, mostly due to loss of functionality from cellular exhaustion. We therefore investigated the phenotypic and functional differences among in vitro activated CD8(+) T cells of three different sources, namely naïve (NTeff), memory (MTeff) and tumor-infiltrating lymphocytes (TILeff) from human and mice, to better understand mechanisms behind potent effector functions and potential for overcoming current limitations. In line with the greater proliferation activity and longer telomere lengths of NTeff populations, cells of naïve origin exhibited significantly less amounts of T cell exhaustion markers than those of MTeff and TILeff, and moreover, acquired distinct expression patterns of memory-promoting transcription factors, T-bet and Eomes, induced in a rapid and sustainable manner. NTeff cells appeared to have lower expression of Foxp1 and were refractory to apoptosis upon TGF-β conditioning, implying better survival potential and resistance to tumor-induced immune suppression. Of CD8(+) T cell pools activated to tumor-specific CTLs, naïve cell generated effectors possessed the most potent cytotoxic activity, validating implications for use in rational design of adoptive immunotherapy.