Medulloblastoma is a highly malignant pediatric brain tumor associated with poor outcome. Developing treatments that target the cancer stem cell (CSC) population in medulloblastoma are important to prevent tumor relapse and induce long-lasting clinical responses. We utilized medulloblastoma neurospheres that display CSC characteristics and found activation of the PI3K/AKT pathway in sphere-forming cells. Of all class IA PI3Ks, only the PI3Kalpha isoform was required for sphere formation by medulloblastoma cells. Knockdown of p110alpha, but not p110beta or p110delta, significantly disrupted cancer stem cell frequencies as determined by extreme limiting dilution analysis (ELDA), indicating an essential role for the PI3Kalpha catalytic isoform in medulloblastoma CSCs. Importantly, pharmacologic inhibition of the MAPK-interacting kinase (MNK) enhanced the antineoplastic effects of targeted PI3Kalpha inhibition in medulloblastoma. This indicates that MNK signaling promotes survival in medulloblastoma, suggesting dual PI3Kalpha and MNK inhibition may provide a novel approach to target and eliminate medulloblastoma CSCs. We also observed a significant reduction in tumor formation in subcutaneous and intracranial mouse xenograft models, which further suggests that this combinatorial approach may represent an efficient therapeutic strategy for medulloblastoma. IMPLICATIONS: These findings raise the possibility of a unique therapeutic approach for medulloblastoma, involving MNK targeting to sensitize medulloblastoma CSCs to PI3Kalpha inhibition.