INTRODUCTION: Stroke is the second most common cause of death in the world and 85% of strokes are ischemic due to obstruction of blood vessels. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. There is an increasing interest in developing new therapies for ischemic stroke due to short therapeutic window and lack of currently available treatments. Astrocytes provide a major role in brain’s response to ischemic injury by providing antioxidant protection, growth factors and glutamate clearance. A detailed understanding of astrocyte-neuron interaction can provide valuable therapeutic targets. This study aims to use astroglial-neuronal co-cultures to investigate affect of hypoxia on interaction between astrocytes and neurons. ----- METHODS: Primary astrocytes were obtained from mouse newborns and NSCs from E14.5 mouse embryos. Astrocytes and NSCs were seeded together to develop astroglial-neuronal co-cultures, which were incubated in normoxic and hypoxic conditions. Co-cultures were characterized using live imagining and immunocytochemistry, quantification of cellular motility and relative gene expression of apoptotic (caspase 8) and necroptotic marker (MLKL) was performed using live cell tracing and rt-PCR respectively. ----- RESULTS: The characterization of co-cultures in hypoxic conditions showed NSCs differentiation into unipolar and bipolar neurons and presence of flat and epithelioid astrocytes. The average and maximum speed of cells in astroglial-neuronal co-cultures was higher in hypoxic conditions, but less than NSCs single culture in normoxia. Relative gene expression of caspase 8 was higher than MLKL in both hypoxic and normoxic conditions. ----- DISCUSSION: There was better differentiation and survival of NSCs in astroglial-neuronal co-cultures in hypoxic conditions. This was also reflected in better cellular motility of astroglialneuronal co-cultures in hypoxic conditions. Caspase 8 and MLKL exhibited reciprocal relationship of expression in both normoxic and hypoxic conditions. Although we observed difference in expression of caspase 8 and MLKL in normoxic and hypoxic conditions, more samples will be needed to define precise levels of statistical significance of this finding. ----- CONCLUSION: In this work we observed responses of astrocytes and neurons in astroglialneuronal co-cultures, which were fairly consistent with what happens in ischemia. More studies will be required to characterize and understand observed phenomena in the astroglialneuronal co-cultures.