Simple SummaryGlioblastoma is among the most lethal cancers in humans. Glioblastomas exhibit a striking heterogeneity, which is maintained by a highly plastic, adaptable population of glioma stem cells (GSCs). Glioblastomas inevitably recur as therapy-resilient tumors, and accumulating evidence indicates key roles for GSCs in recurrence and resistance. GSCs contribute to therapy resistance not only by promoting tumor heterogeneity, but also by modulating components of the tumor environment. This includes crosstalk with cells of the immune system, facilitating immune evasion of malignant cells. This review revisits the decades-long journey from the identification and characterization of GSCs to elucidating their contributions to glioblastoma malignancy and mechanisms of therapy resistance. Finally, the most recent insights in GSC biology are discussed, including novel strategies that specifically target GSCs in the age of immune-based strategies.AbstractSince their discovery at the beginning of this millennium, glioma stem cells (GSCs) have sparked extensive research and an energetic scientific debate about their contribution to glioblastoma (GBM) initiation, progression, relapse, and resistance. Different molecular subtypes of GBM coexist within the same tumor, and they display differential sensitivity to chemotherapy. GSCs contribute to tumor heterogeneity and recapitulate pathway alterations described for the three GBM subtypes found in patients. GSCs show a high degree of plasticity, allowing for interconversion between different molecular GBM subtypes, with distinct proliferative potential, and different degrees of self-renewal and differentiation. This high degree of plasticity permits adaptation to the environmental changes introduced by chemo- and radiation therapy. Evidence from mouse models indicates that GSCs repopulate brain tumors after therapeutic intervention, and due to GSC plasticity, they reconstitute heterogeneity in recurrent tumors. GSCs are also inherently resilient to standard-of-care therapy, and mechanisms of resistance include enhanced DNA damage repair, MGMT promoter demethylation, autophagy, impaired induction of apoptosis, metabolic adaptation, chemoresistance, and immune evasion. The remarkable oncogenic properties of GSCs have inspired considerable interest in better understanding GSC biology and functions, as they might represent attractive targets to advance the currently limited therapeutic options for GBM patients. This has raised expectations for the development of novel targeted therapeutic approaches, including targeting GSC plasticity, chimeric antigen receptor T (CAR T) cells, and oncolytic viruses. In this review, we focus on the role of GSCs as drivers of GBM and therapy resistance, and we discuss how insights into GSC biology and plasticity might advance GSC-directed curative approaches.