Stimulator of interferon genes (STING) signaling induces IFNβ production by intratumoral dendritic cells (DC), driving T-cell priming and recruitment into the tumor microenvironment (TME). We examined to what extent preexisting antigen-specific tolerance influenced the efficacy of in situ delivery of a potent STING-activating cyclic dinucleotide (CDN), ADU S-100, against established HER-2⁺ breast tumors. ADU S-100 induced HER-2–specific CD8⁺ T-cell priming and durable tumor clearance in 100% of nontolerant parental FVB/N mice. In contrast, ADU S-100 did not sufficiently prime HER-2–specific CD8⁺ T cells in tolerant neu/N mice, resulting in only delayed tumor growth and tumor clearance in 10% of the mice. No differences in IFNβ production, DC priming, or HER-2–specific CD8⁺ T-cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2–specific CD8⁺ T cells were defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high numbers of PD1 and OX40 receptors on their CD8⁺ T cells, and PD-L1 was highly expressed on both myeloid and tumor cells. Modulating PD-L1 and OX40 receptor signaling combined with intratumoral ADU S-100 administration enhanced HER-2–specific CD8⁺ T-cell activity, clearing tumors in 40% of neu/N mice. Thus, intratumoral STING agonists could potently prime tumor antigen–specific CD8⁺ T-cell responses, and adding PD-L1 blockade and OX40 receptor activation can overcome antigen-enforced immune tolerance to induce tumor regression. Cancer Immunol Res; 5(6); 468–79. ©2017 AACR.