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Introduction: Cancer stem cells (CSCs) represent a distinct cellular population that has been implicated in tumor recurrence following chemotherapy. Accumulating evidence indicates that BMP signaling plays an important role in the regulation of CSCs, and may be involved in the resistance of CSCs to cytotoxic drugs. The role of BMP pathway regulation of the CSC phenotype responsible for treatment resistance in glioblastoma (GBM) has not been explored. Methods: Paraffin-embedded (FFPE) samples from GBM patients were used to evaluate the expression of markers for BMP signaling (pSMAD1/5/8), proliferation (PCNA), and stemness (SOX2) by immunostaining. We tested the self-renewal capacity of primary patient-derived glioma stem cell (GSC) lines using a sphere formation assay. Expression of stemness markers SOX2 and Bmi-1 in BMP4 treated and untreated GSCs, was analyzed by Western blotting. We also performed xenograft transplantation, whereby GSCs were injected intracranially into immunedeficient mice. When tumor formation was confirmed post-injection, mice were administrated EdU and sacrificed at successive time points thereafter. The frequency of all EdU positive cells as well as ID-1positive/ EdU positive cells was quantified from the primary tumor sections throughout the labeling and chase. To study chemoresistance in glioblastoma, GSC lines were treated with graded concentrations of temozolomide (TMZ). Results: We found that pSMAD1/5/8 positive cells in primary patient tumors were largely PCNA-negative. BMP4 treatment inhibited GSC proliferation and self-renewal (sphere-formation), but did not abolish expression of markers of stemness, such as Sox2 and Bmi-1, or tumorigenicity. In our in vivo model, we found ID-1 positive cells retain EdU following a long-term chase. Taken together, these results demonstrate that BMP4-enriched cells possess the characteristics of a quiescent stem cell population. BMP4 treatment protects GSCs against TMZ-induced growth inhibition. Furthermore, pSMAD1/5/8-expressing cells do not possess the DNA damage marker gamma-H2AX following TMZ treatment, suggesting that activation of the BMP pathway protects GSCs against TMZ-induced DNA damage. Conclusion: This study establishes that the BMP signaling pathway is involved in the maintenance of GSC quiescence and may play a role in glioblastoma chemoresistance. Citation Format: Megan YiJun Wu, Angela Celebre, Jeffrey Chan, Jennifer Guan, Karan Dhand, James Loukides, Jason Karamchandani, Sunit Das. BMP regulation of cancer stem cell quiescence is responsible for chemotherapeutic resistance in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B35.