Malignant astrocytomas of the brainstem and cerebral hemispheres have a poor prognosis despite current treatments, and new therapeutic approaches are needed. Based on our findings regarding antigen expression profiles of childhood gliomas, we initiated a pilot trial of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes emulsified in Montanide-ISA-51 every 3 weeks for 8 courses, and intramuscular administration of poly-ICLC in HLA-A2+ children with newly diagnosed brainstem glioma (BSG), cerebral high-grade glioma (HGG), or recurrent glioma. GAAs were EphA2, interleukin (IL)-13 receptor-α2, and survivin. Primary endpoints were safety and T cell responses against vaccine-targeted GAAs, assessed by ELISPOT and tetramer analysis. Treatment response was evaluated clinically and by MRI. To date, 18 children have been enrolled: 10 with newly diagnosed BSGs treated with irradiation, 4 with newly diagnosed HGGs treated with irradiation and concurrent chemotherapy, and 4 with recurrent gliomas. No dose-limiting toxicity has been encountered. One child with a BSG had transient tumor enlargement 4 months after beginning vaccination (7 months post-irradiation) that later regressed and culminated in a sustained partial response (PR), consistent with pseudoprogression. Two other children with BSG who had pseudoprogression and subsequent stabilization also remain alive > 1 year from diagnosis without further intervention. Principal toxicities have included injection site reactions and low-grade fevers, which have been mild. Among 16 patients evaluable for response, 12 had sustained stable disease, 1 had a PR, and 1 has a continuing complete response after surgery. Seven of 10 BSG patients have survived > 11 months after diagnosis. ELISPOT analysis, completed in 5 children to date, showed response to IL13Rα2 (n = 4), EphA2 (n = 2), and survivin (n = 1). Tetramer responses to IL13Rα2 and EphA2 were also noted. Our preliminary results demonstrate that a multipeptide vaccination approach in children with glioma is well tolerated and has evidence of immunological and clinical activity.