The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell‐types involved in CXCL10/CXCR3 signaling under pathological conditions are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N‐methyl‐D‐aspartic acid (NMDA)‐induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes express CXCL10 in response to excitotoxicity. Experiments in OHSCs derived from CXCL10‐deficient (CXCL10^−/−) and CXCR3‐deficient (CXCR3^−/−) revealed that in the absence of CXCL10 or CXCR3, neuronal cell death in the CA1 and CA3 regions was diminished after NMDA‐treatment when compared to wild type OHSCs. In contrast, neuronal cell death in the DG region was enhanced in both CXCL10^−/− and CXCR3^−/− OHSCs in response to a high (50 μM) NMDA‐concentration. Moreover, we show that in the absence of microglia the differential changes in neuronal vulnerability between CXCR3^−/− and wild type OHSCs are fully abrogated and therefore a prominent role for microglia in this process is suggested. Taken together, our results identify a region‐specific role for CXCL10/CXCR3 signaling in neuron‐glia and glia‐glia interactions under pathological conditions. © 2010 Wiley‐Liss, Inc.