Glioblastoma (GBM) is characterized by extensive intratumoral heterogeneity and a complex tumor microenvironment that complicate the preclinical evaluation of gene therapy vectors. We investigated how culture model, epidermal growth factor (EGF) supplementation, and adeno-associated virus (AAV) serotype influence vector-mediated gene delivery in patient-derived GBM models. Patient-derived neurospheres (PDNS) and patient-derived tissue slice cultures (PDTC) were transduced with AAV2 or AAV6 vectors encoding green fluorescent protein (GFP). Transduction was evaluated by live confocal imaging, quantitative PCR, flow cytometry, and immunofluorescence under EGF-containing and EGF-free culture conditions. In PDNS, AAV6 produced significantly greater GFP expression than AAV2 and demonstrated a dose-dependent increase in transduction at both 2 and 5 days after vector exposure. EGF supplementation altered transduction patterns and was associated with changes in PDNS growth and marker-defined stem-like cell populations. In contrast, PDTC preserved tissue architecture and stromal, vascular, and immune-associated compartments while revealing substantial interpatient variability in AAV-mediated transduction. AAV-mediated gene delivery in GBM is influenced by culture model, growth factor conditions, and capsid serotype. Patient-derived tissue slice cultures complement neurosphere models by preserving features of the native tumor microenvironment and may improve the preclinical evaluation of gene therapy vectors for GBM.