ABSTRACT: While blockade of PD-1/PD-L1 immune checkpoint revolutionized cancer treatment, how it works on tumor-infiltrating CD8+ T cells recognizing the same antigen at various differentiation stages remains elusive. Here, we found that the chemokine receptor CX3CR1 identified three distinct differentiation states of intratumoral CD8+ T-cell subsets. Adoptively transferred antigen-specific CX3CR1neg CD8+ T cells generated phenotypically and functionally distinct CX3CR1int and CX3CR1hi subsets in the periphery. Notably, expression of co-inhibitory receptors and Tcf1 inversely correlated with the degree of T-cell differentiation defined by CX3CR1. Despite significantly lower expression of co-inhibitory receptors and potent cytolytic activity, in vivo depletion of the CX3CR1hi subset did not alter the antitumor efficacy of adoptively transferred CD8+ T cells. Furthermore, differentiated CX3CR1int and CX3CR1hi subsets were impaired in their ability to undergo proliferation upon re-stimulation, and had no impact on established tumors upon second adoptive transfer compared with the CX3CR1neg subset that remained effective. Accordingly, anti-PD-L1 therapy preferentially rescued proliferation and cytokine production of the CX3CR1neg subset, and significantly enhanced antitumor efficacy of adoptively transferred CD8+ T cells. These findings provide a better understanding of the phenotypic and functional heterogeneity of tumor-infiltrating CD8+ T cells, and can be exploited to develop more effective immunotherapy.