© 2016 Elsevier Inc.. Activation of nuclear factor κB (NF-κB) induces mesenchymal (MES) transdifferentiation and radioresistance in glioma stem cells (GSCs), but molecular mechanisms for NF-κB activation in GSCs are currently unknown. Here, we report that mixed lineage kinase 4 (MLK4) is overexpressed in MES but not proneural (PN) GSCs. Silencing MLK4 suppresses self-renewal, motility, tumorigenesis, and radioresistance of MES GSCs via a loss of the MES signature. MLK4 binds and phosphorylates the NF-κB regulator IKKα, leading to activation of NF-κB signaling in GSCs. MLK4 expression is inversely correlated with patient prognosis in MES, but not PN high-grade gliomas. Collectively, our results uncover MLK4 as an upstream regulator of NF-κB signaling and a potential molecular target for the MES subtype of glioblastomas. Kim et al. show that stem-like cells of mesenchymal (MES) glioblastoma (GSCs) overexpress MLK4 and that silencing MLK4 reduces GSCs self-renewal, radioresistance, and tumorigenicity. IKKα is a MLK4 substrate and targeting the MLK4-driven NF-κB signaling could be a therapeutic strategy for MES glioblastoma.