Glioblastomas (GBM) contain numerous hypoxic foci associated with a rare fraction of glioma stem cells (GSCs). Left-right determination factor (LEFTY) and Nodal, members of the transforming growth factor β (TGF-β) superfamily, have glycogen synthase kinase 3β (GSK-3β) phosphorylation motifs and are linked with stemness in human malignancies. Herein, we investigated the roles of LEFTY and Nodal in GBM hypoxic foci. In clinical samples, significantly higher expression of LEFTY, Nodal, phospho (p) GSK-3β, pSmad2, and Nestin, as well as higher apoptotic and lower proliferation rates, were observed in nonpseudopalisading (non-Ps) perinecrotic lesions as compared to Ps and non-necrotic tumor lesions, with a positive correlation between LEFTY, Nodal, pGSK-3β, or pSmad2 scores. In KS-1, a GBM cell line that lacks endogenous Nodal expression, treatment with the hypoxic mimetic CoCl 2 increased LEFTY, pGSK-3β, and pSmad2 levels, but decreased pAkt levels. Moreover, the promoter for LEFTY, but not Nodal, was activated by Smad2 or TGF-β1, suggesting that overexpression of LEFTY and Nodal may be due to Akt-independent GSK-3β inactivation, with or without cooperation of the TGF-β1/Smad2 axis. LEFTY and Nodal overexpression increased proliferation rates and reduced susceptibility to CoCl 2-induced apoptosis, and increased the expression of epithelialmesenchymal transition (EMT)/GSC-related markers. An increased ALDH1 high population and more efficient spheroid formation was also observed in LEFTY-overexpressing cells. These findings suggest that LEFTY and Nodal may contribute to cell survival in non-Ps GBM perinecrotic lesions, leading to alterations in apoptosis, proliferation, or EMT/GCS features.