We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through amechanism associated with intracellularGSHdepletionmediated by the bidirectional cystine/Glu antiporter in osteoblasticMC3T3-E1 cells cultured in the absence of differentiation inducers. To further evaluate the possible role of the antiporter in osteoblastogenesis, in this study, we have established stable transfectants of the xCT subunit of the antiporter in MC3T3-E1 cells. Stable overexpression led to a significant facilitation of cellular proliferation determined by different indices with increased GSH levels and decreased ROS generation in addition to promoted [(14)C] cystine incorporation, while Glu failed to significantly inhibit cellular proliferation in stable xCT transfectants. In stable transfectants cultured under differentiation conditions, drastic decreases were invariably seen in Ca(2+) accumulation, alkaline phosphatase activity and several osteoblastic marker gene expressions, in addition to downregulation ofmRNA and corresponding protein for runt-related transcription factor-2 (Runx2). Runx2 promoter activitywas significantly promoted by the introduction ofRunx2 expression vector in amanner sensitive to the prevention by the co-introduction of xCT expression vector in MC3T3-E1 cells. In both MC3T3-E1 cells and murine calvarial osteoblasts cultured with differentiation inducers, transient transfection with xCT siRNA significantly increased Runx2 protein expression alongwith decreases in xCT mRNA expression and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction. These results suggest that the cystine/ Glu antiporter plays a pivotal role in cellular differentiation through a mechanismrelated to the regulation of transactivation of Runx2 essential for osteoblastogenesis toward maturation in osteoblastic cells. J. Cell. Physiol. 226: 2953-2964, 2011. (C) 2011 Wiley-Liss, Inc.