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Summary: Glioblastoma (GBM) is the most common malignant adult brain tumor that is resistant to the standard care therapy. Advances in chimeric antigen receptor (CAR) T cell therapies have spurred renewed interest in developing CAR T cell therapies to target chemoradiotherapy-resistant brain tumor-initiating cells. This protocol shows how to isolate peripheral blood mononuclear cells from healthy donors and generate CAR T cells for the antigens of interest, and how to intracranially inject the CAR T cells into a patient-derived xenograft mouse model of GBM.For complete details on the use and execution of this protocol, please refer to Vora et al. (2020).