Immunotherapy for cancer treatment requires the activation of cytotoxic effector lymphocytes. Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize the MHC class I–like molecule MR1. MAIT cells play an important role in the immune response against microbial infections and can directly kill tumor cells. Although MAIT cells can be expanded ex vivo, this method is time-consuming, expensive, and requires allogenic feeder layers. To overcome the limitations of conventional dendritic cell–based vaccines and ex vivo expansion of human T cells, an artificial APC (aAPC) approach to expand antitumor effector cells has several advantages. In this study, we explored an efficient in vitro method to amplify MR1-specific MAIT cells from human peripheral blood using aAPCs made by coating cell-sized latex beads with an Ag-loaded MR1 tetramer complex and anti-CD28 Ab. We further elucidated the cytotoxic potential of such expanded MAIT cells against three human glioblastoma multiforme (GBM) cell lines to explore their potential use as a novel immunotherapeutic tool, as the mostly lethal GBM poorly responds to conventional chemotherapy. When aAPCs were compared with the standard allogenic feeder layer–based approach for MAIT cell expansion, they were significantly more effective. Our results indicate that the aAPC-expanded MAIT cells remained functional, retained their original phenotype, secreted proinflammatory cytokines, and showed cytotoxicity against the GBM cell lines. Hence, MAIT cells have the potential to be a novel tool in immunotherapy approaches for the treatment of human GBM.