Mesenchymal stromal cells (MSCs) are multipotent post-natal stem cells with applications in tissue engineering and regenerative medicine. MSCs can differentiate into osteoblasts, chondrocytes, or adipocytes, with functional differences in cells during osteogenesis accompanied by metabolic changes. The temporal dynamics of these metabolic shifts have not yet been fully characterized and are suspected to be important for therapeutic applications such as osteogenesis optimization. Here, our goal was to characterize the metabolic shifts that occur during osteogenesis. We profiled five key extracellular metabolites longitudinally (glucose, lactate, glutamine, glutamate, and ammonia) from MSCs from four donors to classify osteogenic differentiation into three metabolic stages, defined by changes in the uptake and secretion rates of the metabolites in cell culture media. We used a combination of untargeted metabolomic analysis, targeted analysis of 13C-glucose labelled intracellular data, and RNA-sequencing data to reconstruct a gene regulatory network and further characterize cellular metabolism. The metabolic stages identified in this proof-of-concept study provide a framework for more detailed investigations aimed at identifying biomarkers of osteogenic differentiation and small molecule interventions to optimize MSC differentiation for clinical applications.