AbstractTherapies that target the signal transduction and biological characteristics of cancer stem cells (CSCs) are innovative strategies that are used in combination with conventional chemotherapy and radiotherapy to effectively reduce the recurrence and significantly improve the treatment of glioblastoma multiforme (GBM). The two main strategies that are currently being exploited to eradicate CSCs are (a) chemotherapeutic regimens that specifically drive CSCs toward cell death and (b) those that promote the differentiation of CSCs, thereby depleting the tumour reservoir. Extracellular purines, particularly adenosine triphosphate, have been implicated in the regulation of CSC formation, but currently, no data on the role of adenosine and its receptors in the biological processes of CSCs are available. In this study, we investigated the role of adenosine receptor (AR) subtypes in the survival and differentiation of CSCs isolated from human GBM cells. Stimulation of A₁AR and A_2BAR had a prominent anti-proliferative/pro-apoptotic effect on the CSCs. Notably, an A₁AR agonist also promoted the differentiation of CSCs toward a glial phenotype. The differential effects of the two AR agonists on the survival and/or differentiation of CSCs may be ascribed to their distinct regulation of the kinetics of ERK/AKT phosphorylation and the expression of hypoxia-inducible factors. Most importantly, the AR agonists sensitised CSCs to the genotoxic activity of temozolomide (TMZ) and prolonged its effects, most likely through different mechanisms, are as follows: (i) by A_2BAR potentiating the pro-apoptotic effects of TMZ and (ii) by A₁AR driving cells toward a differentiated phenotype that is more sensitive to TMZ. Taken together, the results of this study suggested that the purinergic system is a novel target for a stem cell-oriented therapy that could reduce the recurrence of GBM and improve the survival rate of GBM patients.