We have shown that glutamate (Glu) signaling machineries, such as receptors (GluR) and transporters, are functionally expressed by mesenchymal stem cells, in addition to by their progeny cells such as osteoblasts and chondrocytes. Sustained exposure to Glu induced significant decreases in alkaline phosphatase (ALP) staining and osteoblastic marker gene expression in the mesenchymal C3H10T1/2 stem cells infected with runt-related transcription factor-2 (Runx2) adenovirus, without markedly affecting Oil Red O staining for adipocytes in cells cultured with adipogenic inducers. In cells with Runx2 adenovirus, the cystine/Glu antiporter substrate cystine significantly prevented the decreases by Glu in both ALP staining and osteoblastic marker gene expression, with GluR agonists being ineffective. In cells with Runx2 adenovirus, Glu significantly decreased [C-14] cystine uptake, intracellular glutathione (GSH) level, Runx2 recruitment to osteocalcin promoter and nuclear Runx2 protein level, respectively. Cystine again significantly prevented the decreases by Glu in both GSH levels and Runx2 recruitment. In mouse bone marrow stromal cells, Glu and a GSH depleter significantly decreased ALP staining without affecting Oil Red O staining. Knockdown of the cystine/Glu antiporter led to markedly decreased ALP staining and GSH levels, with concomitant prevention of the decrease by Glu, in cells with Runx2 adenovirus. These results suggest that Glu may play a role as a negative regulator at an early differentiation stage into osteoblasts than adipocytes through a mechanism relevant to nuclear translocation of Runx2 after regulation of intracellular GSH levels by the cystine/Glu antiporter expressed in mesenchymal stem cells. J. Cell. Physiol. 226: 652-665, 2011. (C) 2010 Wiley-Liss, Inc.