RELA-fused supratentorial (ST) ependymoma (EPN) is an aggressive subgroup with poor prognosis. Considering the putative role of Notch signaling in the maintenance of the cancer stem cells (CSC) phenotype in RELA-fused EPN, we investigated the expression of Notch pathway and its target genes in this subgroup. We also evaluated the effects of two Notch inhibitors (DAPT and RO4929097) on cell proliferation, apoptosis, colony formation, and CSCs markers gene expression on EPN cell line of the RELA-fused subgroup (BXD-1425). In addition, in silico signatures of the Notch genes and CSCs markers were analyzed on a large clinical dataset from GSE64415 study. We found that among the ST-EPN subgroups the Notch signaling (NOTCH1, JAG1, JAG2, and HES4) is specifically activated in the ST-EPN-RELA. Furthermore, treatment of the RELA-fused EPN cell line with the Notch inhibitors impaired the Notch signaling expression and revealed that Notch axis is not essential for cell proliferation and survival in this setting. NOTCH1 expression in ST-EPN was correlated with the CSCs markers VEGFA and L1CAM overexpression and JAG1 expression was correlated with the CCND1 and CDK6 overexpression. In addition, in vitro treatment with Notch inhibitors induced downregulation of CSCs markers. These findings indicate that Notch signaling can be involved in the ST-EPN-RELA CSCs maintenance by modulating the expression of genes responsible for cell phenotype and cell fate.