Mesenchymal stromal cells (MSCs) are early progenitors that can differentiate into osteoblasts, chondrocytes and adipocytes. We hypothesized that osteoblasts and adipocytes utilize distinct bioenergetic pathways during MSC differentiation. To test this hypothesis, we compared the bioenergetic profiles of pre-osteoblast MC3T3-E1 cells and calvarial osteoblasts with pre-adipocyte 3T3L1 cells, before and after differentiation. Differentiated MC3T3-E1 osteoblasts met ATP demand mainly by glycolysis with minimal reserve glycolytic capacity, whereas non-differentiated cells generated ATP through oxidative phosphorylation. A marked Crabtree effect (acute suppression of respiration by addition of glucose, observed in both MC3T3-E1 and calvarial osteoblasts) and smaller mitochondrial membrane potential in the differentiated osteoblasts, particularly those incubated at high glucose concentrations, indicated a suppression of oxidative phosphorylation compared to non-differentiated osteoblasts. In contrast, both non-differentiated and differentiated 3T3-L1 adipocytes met ATP demand primarily by oxidative phosphorylation despite a large unused reserve glycolytic capacity. In sum, we show that non-differentiated precursor cells prefer to use oxidative phosphorylation to generate ATP; when they differentiate to osteoblasts they gain a strong preference for glycolytic ATP generation, but when they differentiate to adipocytes they retain the strong preference for oxidative phosphorylation. Unique metabolic programming in mesenchymal progenitor cells may influence cell fate and ultimately determine the degree of bone formation and/or the development of marrow adiposity. This article is protected by copyright. All rights reserved.