Glioblastoma remains among the most aggressive of all human and canine malignancies, displaying high mortality rates and limited treatment options. We propose that given the similarities between canine and human gliomas, such as incidence of occurrence, histopathology, molecular characteristics, and response to therapy, that canine gliomas are a natural model of the human disease. A range of human and canine tumours have been shown to harbor specific subpopulations of cells with stem cell-like properties that initiate and maintain neoplasticity while resisting conventional therapies. Here, we show that both canine and human glioma cell lines contain a small population of cancer stem cells (CSCs), and by molecular profiling highlight the important role of the epidermal growth factor receptor (EGFR) pathway in canine CSCs. EGFR signaling is crucial in the regulation of cancer cell proliferation, migration and survival. To date EGFR-targeted interventions alone have been largely ineffective. Our findings confirm that specifically inhibiting EGFR signaling alone has no significant effect on the viability of CSCs. However inhibition of EGFR did enhance the chemo- and radio-sensitivity of both canine and human glioma CSCs, enabling this resistant, tumourigenic population of cells to be effectively targeted by conventional therapies.