Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca2+ ([Ca2+](o) and [Ca2+](i)) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca2+](o) and high [Ca2+](i) enhanced adipocyte accumulation, which suggested that increases in [Ca2+](o) caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca2+](o), and high [Ca2+](i) may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MU assay and real-time RT-PCR revealed that high [Ca2+](i) (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca2+](o) (addition of CaCl2) leads to increases in [Ca2+](i). Flow cytometric methods revealed that high [Ca2+](o) suppressed the phosphorylation of ERK independently of intracellular Ca2+. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca2+ provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca2+, which results in BMSC proliferation. (C) 2015 Elsevier Inc. All rights reserved.