Glioblastoma multiforme (GBM) is one of the most aggressive brain tumors. GBM cell lines used in laboratory studies are frequently passaged in various culture media at high proliferation rates, resulting in significant genetic and molecular alterations. Thus, data obtained in cell lines are often inapplicable to patient tumors. Furthermore, recent studies suggest that there is a stem cell-like hierarchy among GBM cell populations and a crucial role for tumor vasculature in stem cells, as well as tumor growth, which cannot be reproduced in cell line cultures. Our laboratory has developed a novel three-dimensional (3D) organotypic "explant" system of surgical GBM specimens that preserves tumor cells in their original milieu, as well as the cytoarchitecture of the tumor stroma. Our previous study on the role of Notch inhibition has demonstrated a definitive effect on the tumor endothelium that could only be highlighted by this system. In this unit, we describe a detailed protocol for preparing GBM explants, and discuss strengths, as well as limitations of the explant system as an in vitro 3D model of GBM.