Median survival for glioblastoma (GBM) remains <15 months. Human Cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However, ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DC would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here we report prospective results of a pilot trial in which 22 patients with newly-diagnosed GBM were initially enrolled of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-Saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNgamma+, TNFalpha+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.