Radiation therapy has the potential to augment immune responses against central nervous system tumors. Furthermore, cancer cells destroyed by radiation therapy are considered to be a source of tumor associated antigens that can be processed by professional antigen presenting cells. We investigated the use of focal radiation therapy in addition to anti-4-1BB and Kojic acid anti-CTLA-4 immunotherapy as a combination strategy in an orthotopic, preclinical model of malignant glioma. We hypothesized that radiation therapy followed by 4-1BB activation and CTLA-4 blockade produces an effective and durable anti-tumor response against intracranial GL261 gliomas. The orthotopic glioma model was established as previously described. Animals were stratified into treatment groups on day 7 following intracranial implantation based on tumor burden as determined by bioluminescent imaging. For those mice treated with focal radiation therapy, a Fenobam single fraction of radiation at a dose of 10 Gy was delivered using a 3 mm collimator on day 10 following intracranial implantation using the Small Animal Radiation Research Platform. With the built-in micro-CT scanner we identified the burr hole, which served as the coordinate for delivery of radiation. In those mice treated with anti-4-1BB mAb, 200 mg of anti-4-1BB antibodies was dosed via intra-peritoneal injection on days 11, 14 and 17 following intracranial implantation. In mice treated with anti-CTLA-4 mAb, 800 mg of anti-CTLA-4 antibodies was dosed via intra-peritoneal injection on days 11, 17 and 23. Controls in all treatment groups received rat and hamster IgG. For the pilot experiments in which treatment with a single antibody was compared to combination therapy with SRS and antibody therapy, five animals were used per treatment group and this experiment was performed once. In the CTLA-4 timing experiments, eight animals were used per treatment group and these experiments were repeated twice. The triple therapy experiments were repeated three times and a total of 18 animals per treatment group were used. Animals were observed three times a week for signs of lethargy such as weight loss, hunched position and epilepsy.