Glioblastoma is the most aggressive and deadly form of primary brain cancer, with no effective treatments available. However, targeted immunotherapies such as chimeric antigen receptor (CAR) T cells represent a potential new treatment approach. To guide the rational development of such therapies, we have conducted a detailed analysis of the tumour and stromal cells in the glioblastoma microenvironment using single cell transcriptomics, high-parameter flow cytometry and tissue immunostaining. Key findings include (i) identification and characterisation of distinct populations of lymphocytes, macrophages, endothelial cells and pericytes within patient tumours; (ii) discovery of a promising CAR-T cell target antigen with expression not only on tumour cells, but also ubiquitous expression on tumour blood vessels; and (iii) detailed profiling of the homing receptors expressed by T cells within the glioblastoma microenvironment. These findings are now guiding our development of novel CAR-T cell therapies for glioblastoma by allowing us to identify optimal targeting approaches (including co-targeting of tumour cells and their supporting vasculature), and to engineer our therapeutic T cells for maximal tumour homing by mimicking the cues that guide endogenous T cells into tumours.