Abstract To identify new candidate therapeutic targets for glioblastoma multiforme, we combined functional genetics and glioblastoma network modeling to identify kinases required for the growth of patient-derived brain tumor–initiating cells (BTIC) but that are dispensable to proliferating human neural stem cells (NSC). This approach yielded BUB1B/BUBR1, a critical mitotic spindle checkpoint player, as the top-scoring glioblastoma lethal kinase. Knockdown of BUB1B inhibited expansion of BTIC isolates, both in vitro and in vivo, without affecting proliferation of NSCs or astrocytes. Mechanistic studies revealed that BUB1B's GLE2p-binding sequence (GLEBS) domain activity is required to suppress lethal kinetochore–microtubule (KT–MT) attachment defects in glioblastoma isolates and genetically transformed cells with altered sister KT dynamics, which likely favor KT–MT instability. These results indicate that glioblastoma tumors have an added requirement for BUB1B to suppress lethal consequences of altered KT function and further suggest that sister KT measurements may predict cancer-specific sensitivity to BUB1B inhibition and perhaps other mitotic targets that affect KT–MT stability. Significance: Currently, no effective therapies are available for glioblastoma, the most frequent and aggressive brain tumor. Our results suggest that targeting the GLEBS domain activity of BUB1B may provide a therapeutic window for glioblastoma, as the GLEBS domain is nonessential in untransformed cells. Moreover, the results further suggest that sister KT distances at metaphase may predict sensitivity to anticancer therapeutics targeting KT function. Cancer Discov; 3(2); 198–211. ©2012 AACR. See related commentary by Venere et al., p. 141 This article is highlighted in the In This Issue feature, p. 125