ARTICLE ABSTRACTGlioblastoma (GBM)–initiating cells (GIC) are a tumorigenic subpopulation that are resistant to radio- and chemotherapies and are the source of disease recurrence. Therefore, the identification and characterization of GIC-specific factors is critical toward the generation of effective GBM therapeutics. In this study, we investigated the role of epithelial V-like antigen 1 (Eva1, also known as myelin protein zero-like 2) in stemness and GBM tumorigenesis. Eva1 was prominently expressed in GICs in vitro and in stem cell marker (Sox2, CD15, CD49f)-expressing cells derived from human GBM tissues. Eva1 knockdown in GICs reduced their self-renewal and tumor-forming capabilities, whereas Eva1 overexpression enhanced these properties. Eva1 deficiency was also associated with decreased expression of stemness-related genes, indicating a requirement for Eva1 in maintaining GIC pluripotency. We further demonstrate that Eva1 induced GIC proliferation through the activation of the RelB-dependent noncanonical NF-κB pathway by recruiting TRAF2 to the cytoplasmic tail. Taken together, our findings highlight Eva1 as a novel regulator of GIC function and also provide new mechanistic insight into the role of noncanonical NF-κB activation in GIC, thus offering multiple potential therapeutic targets for preclinical investigation in GBM. Cancer Res; 76(1); 171–81. ©2015 AACR.