Memory CD8+ T cells have long been considered a promising population for adoptive cell therapy (ACT) due to their long-term persistence and robust re-stimulatory response. NIH3T3 is an immortalized mouse embryonic fibroblast cell line. We report that NIH3T3-conditioned medium (CM) can augment effector functions of CTLs following antigen priming and confer phenotypic and transcriptional properties of central memory cells. After NIH3T3-CM-educated CTLs were infused into na?ve mice, they predominantly developed to central memory cells. A large number of NIH3T3-CM-educated CTLs with high functionality persisted and infiltrated to tumor mass. In addition, NIH3T3-CM inhibited CTLs expression of PD-1 in vitro and repressed their high expression of PD-1 in tumor microenvironment after adoptive transfer. Consequently, established tumor models showed that infusion of NIH3T3-CM-educated CTLs dramatically improved CTL mediated-antitumor immunity. Furthermore, NIH3T3-CM also promoted human CD8+ T cells differentiation into memory cells. These results suggest that NIH3T3-CM-programmed CTLs are good candidates for adoptive transfer in tumor therapy.