Stem cells reside in niches that regulate the balance between self-renewal and differentiation. The identity of a stem cell is linked with the ability tointeract with its niche through adhesion mechanisms. To identify targets that disrupt cancer stemcell (CSC) adhesion, we performed a flow cytometryscreen on patient-derived glioblastoma (GBM) cells and identified junctional adhesion molecule A(JAM-A) as a CSC adhesion mechanism essentialfor self-renewal and tumor growth. JAM-A wasdispensable for normal neural stem/progenitorcell (NPC) function, and JAM-A expression was reduced in normal brain versus GBM. Targeting JAM-A compromised the self-renewal of CSCs. JAM-A expression negatively correlated to GBM patient prognosis. Our results demonstrate thatGBM-targeting strategies can be identified through screening adhesion receptors and JAM-A represents a mechanism for niche-driven CSC maintenance. © 2014 The Authors.