Abstract Glioblastomas (GBMs), the most malignant glial tumors, differ from normal glial progenitor cells in many ways, but little is known about the molecular circuitry underlying these differences. We have discovered a novel means by which GBMs become chemo-resistant, based on inhibition of the redox/Fyn/c-Cbl pathway by overexpression of Cool-1. The protein c-Cbl, an E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of multiple receptor tyrosine kinases critical for cell division and cell survival. In normal glial progenitor cells of the CNS, exposure to chemotherapy oxidizes cells, leading to sequential activation of Fyn and c-Cbl. However in GBM cells, exposure to BCNU or other chemical pro-oxidants does not lead to c-Cbl activation. Further studies have demonstrated that these agents do cause Fyn activation, suggesting that GBMs inhibit c-Cbl phosphorylation leading to decreased degradation of EGFR, accounting for increased EGFR signaling. We therefore set out to investigate the factors involved in the decreased c-Cbl activity specific to GBMs. In this work we discovered that oxidant-associated c-Cbl activation can be restored by suppressing expression of the protein Cool-1, found to be over-expressed in GBM. Our data further demonstrate that Cool-1 and c-Cbl formed complexes, that genetic reduction in Cool-1 levels restored normal c-Cbl-mediated degradation of RTKs (including EGFR) in response to exposure to pro-oxidants, and that restoration of normal c-Cbl activation has multiple effects on hallmarks of GBM. Cool-1 decreases lead to decreased migration and decreased numbers of cells in the cell cycle. Cool-1 inhibition leads to decreased number of cells that exhibit cancer stem cells characteristics including decreased antigen positivity and decreased sphere forming ability. In contrast, Cool-1/c-Cbl complexes were not found in normal brain or normal glial progenitors and Cool-1 knockdown did not reduce progenitor division. The translation of these findings into an in vivo intracranial xenograft model of GBM shows that Cool-1 is essential for tumorigenesis and decreasing Cool-1 levels leads to decreased tumor take, decreased tumor growth, and increased survival. In sum, the ability of Cool-1/c-Cbl interactions, to modulate a variety of tumor cell properties suggests that these interactions offer an attractive target for modulating GBM growth, particularly due to the absence of Cool-1/c-Cbl complexes in normal brain tissue and to the lack of effects of Cool-1 knockdown on division of normal glial progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2074. doi:1538-7445.AM2012-2074