Glioma stem cells (GSCs) have been implicated in the promotion of malignant progression. Epidermal growth factor receptor variant (EGFRv) has been associated with glioma "stemness". However, the molecular mechanism is not clear. In this study, we were committed to investigate the role of EGFRv in GSCs and presented a new therapeutic target in EGFRvIII positive GSCs. The results showed that EGFRvIII could induce the expression of p-Src and PLK1, and both could induce the Notch1-SOX2 signaling pathway to promote self-renewal and tumor progression of GSCs. Mechanistically, both p-Src and PLK1 can induce Notch1, and the intracellular domain of Notch1 (NICD) can directly bind to SOX2, thereby promoting the maintenance of glioma stem cells. Furthermore, Saracatinib (Src inhibition) and BI2536 (PLK1 inhibition) diminished GSC self-renewal in vitro, and combining the two inhibitors increased survival of orthotopic tumor-bearing mice. Taken together, these data indicate that p-Src and PLK1 contribute to cancer stemness in EGFRvIII-positive GSCs by driving Notch1-SOX2 signaling, a finding that has important clinical implications.Keywords: EGFRvIII; Glioma stem cells (GSCs); Notch1-SOX2; PLK1; p-Src.