PURPOSE: Immune checkpoint inhibitors (ICI) are used for the treatment of various cancers, but clinical trials of anti-programmed cell death protein 1 (PD-1) with patients with recurrent glioblastoma (GBM) have failed to show clinical benefits. In this study, we examined the differentiation status of CD8 tumor-infiltrating lymphocytes (TIL) from patients with primary GBM and their reinvigoration by ICIs to understand the nature of T-cell exhaustion in GBM. EXPERIMENTAL DESIGN: We isolated TILs from 98 patients with newly diagnosed GBM and examined the expression of immune checkpoint receptors and T-cell transcription factors using flow cytometry. TILs were stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) and their proliferation assessed. RESULTS: CD8 TILs had significantly increased expression of immune checkpoint receptors, including PD-1 and CTLA-4, compared with peripheral blood CD8 T cells. Among CD8 TILs, PD-1 cells exhibited more terminally differentiated phenotypes (i.e., EomesT-bet) than PD-1 cells. These data were confirmed by analyzing NY-ESO-1-specific CD8 TILs. Evaluating the proliferation of CD8 TILs after stimulation with anti-CD3 and anti-PD-1, we found that proliferation inversely correlated with the percentage of EomesT-bet cells among PD-1CD8 TILs. When anti-CTLA-4 was used in combination with anti-PD-1, an additional increase in CD8 TIL proliferation was observed in patients with low percentages of EomesT-bet CD8 TILs, who responded well to anti-PD-1 in assays, but not in patients with high percentages of EomesT-bet CD8 TILs, who did not respond to anti-PD-1. CONCLUSIONS: In primary GBM, the differentiation status of CD8 TILs determines their reinvigoration ability upon ICI treatment.