Up-regulation of STIM1-mediated store-operated Ca2+ entry (SOCE) and Ca2+-dependent NFAT signaling is important for myogenic differentiation. However, the molecular mechanisms for differentiation specific up-regulation of STIM1/SOCE-mediated signaling are poorly understood. This study explored whether functional crosstalk between STIM1 and a member of NFAT transcription factor is important for C2C12 myoblast differentiation. Transient increase of NFATc3 expression was observed in the initial phase of differentiation, and the increased activity of NFATc3 isoform was correlated with up-regulation of STIM1 expression. Overexpression of NFATc3 increased STIM1 expression, SOCE activity, and myotube formation, whereas NFATc3 knockdown showed the opposite effects. Overexpression of STIM1 increased the activity and expression level of NFATc3, and enhanced myotube formation, whereas STIM1 knockdown resulted in the opposite effects. Taken together, our findings suggest that a positive feedback control between STIM1/SOCE and NFATc3 is required for efficient induction and progression of myoblast differentiation. (C) 2012 Elsevier Inc. All rights reserved.