Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by glioblastoma stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphR that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (Cytometry by Time-Of-Flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSC to identify putative rGBM-specific EphR. EphA2 and EphA3 co-expression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EphA2 and EphA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo. Treatment of rGBM with a bispecific antibody (BsAb) against EphA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of Akt and Erk and increased cellular differentiation. In conclusion, we show that EphA2 and EphA3 together mark a GSC population in rGBM and that strategic co-targeting of EphA2 and EphA3 presents a novel and rational therapeutic approach for rGBM.