Our understanding of glioblastoma multiforme (GBM), the most common form of primary brain cancer, has been significantly advanced by recent efforts to characterize the cancer genome using unbiased high-throughput sequencing analyses. While these studies have documented hundreds of mutations, gene copy alterations, and chromosomal abnormalities, only a subset of these alterations are likely to impact tumor initiation or maintenance. Furthermore, genes that are not altered at the genomic level may play essential roles in tumor initiation and maintenance. Identification of these genes is critical for therapeutic development and investigative methodologies that afford insight into biological function. This requirement has largely been fulfilled with the emergence of RNA interference (RNAi) and high-throughput screening technology. In this article, the authors discuss the application of genome-wide, high-throughput RNAi-based genetic screening as a powerful tool for the rapid and cost-effective identification of genes essential for cancer proliferation and survival. They describe how these technologies have been used to identify genes that are themselves selectively lethal to cancer cells, or synthetically lethal with other oncogenic mutations. The article is intended to provide a platform for how RNAi libraries might contribute to uncovering glioma cell vulnerabilities and provide information that is highly complementary to the structural characterization of the glioblastoma genome. The authors emphasize that unbiased, systems-level structural and functional genetic approaches are complementary efforts that should facilitate the identification of genes involved in the pathogenesis of GBM and permit the identification of novel drug targets.