Purpose:We tested the combination of a tumor lysate vaccinewith a panel of costimulatorymolecules to identify an immunotherapeutic approach capable of curing established murine gliomas. Experimental Design: Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide. Results: Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100%depending on the timing, regimen, and combination with temozolomide. Curedmice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine/Fc-OX40L–treated mice had superior tumoricidal function relative to all other groups. Vaccine/Fc-OX40L–treated mice exhibited a significant increase in proliferation of brain-infiltrating CD4 and CD8 T cells, as indicated by Ki67 staining. Fc-OX40L had singleagent activity in transplanted and spontaneous glioma models, and the pattern of inflammatory gene expression in the tumor predicted the degree of therapeutic response. Conclusions: These data show that Fc-OX40L has unique and potent activity against experimental gliomas and warrants further testing. Clin Cancer Res; 1–12. 2012 AACR.