Brain cancers such as glioblastoma (GBM) cause a disproportionate level of morbidity and mortality among cancer patients. Therapeutic advances over the past decade have done little to change this bleak prognosis, helping only a minority of patients. The brain tumor microenvironment (TME) is highly supportive of the tumors. It differs from other peripheral malignancies due to its unique composition, which includes the glial, neural, and immune cell populations. Until recently, the study of the brain TME was limited by the lack of methods to target the different cells in TME. This protocol describes stereotaxic surgery optimized for gene delivery by recombinant adeno-associated viruses or lentiviruses in mice and rats. This method allows the manipulation of gene expression in the TME with excellent spatiotemporal control in specific cells (or a subpopulation of cells). Many aspects of the technique, including its versatility, ease of application, and high reproducibility, make it an attractive approach for studying cellular and circuit functions in the TME.