Notch signaling is altered in many cancers. Our previous findings in primary pediatric ependymoma support a role for NOTCH in glial oncogenesis. The present study evaluates the γ-secretase inhibitor RO4929097 in glial tumor models. The expression of Notch pathway genes was evaluated using real-time RT-PCR in 21 ependymoma and glioma models. NOTCH1 mutations were analyzed by DNA sequencing. RO4929097 activity was evaluated in vitro and in vivo, as a single agent and in combination, in glioma and ependymoma models. Notch pathway genes are overexpressed in ependymomas and gliomas along with FBXW7 downregulation. NOTCH1 mutations in the TAD domain were observed in 20% (2/10) of ependymoma primary cultures. Blocking the Notch pathway with the γ-secretase inhibitor RO4929097 reduced cell density and viability in ependymoma short-term cultures. When combined with chemotherapeutic agents, RO4929097 enhanced temozolomide effects in ependymoma short-term cultures and potentiated the cytotoxicity of etoposide, cisplatinum, and temozolomide in glioma cells. RO4929097, in combined treatment with mTOR inhibition, potentiated cytotoxicity in vitro, but did not enhance antitumor effects in vivo. In contrast, RO4929097 enhanced irradiation effects in glioma and ependymoma xenografts and showed tumor growth inhibition in advanced-stage IGRG121 glioblastoma xenografts. RO4929097-mediated effects were independent of NOTCH1 mutation status or expression levels, but associated with low IL-6 levels. In established glial tumor models, NOTCH inhibition had limited effects as a single agent, but enhanced efficacy when combined with DNA-interfering agents. These preclinical data need to be considered for further clinical development of NOTCH inhibitors in glial tumors.