Skeletal stem cells (SSCs) are postulated to provide a continuous supply of osteoblasts throughout life. However, under certain conditions, the SSC population can become incorrectly specified or is not maintained, resulting in reduced osteoblast formation, decreased bone mass, and in severe cases, osteoporosis. Glutamine metabolism has emerged as a critical regulator of many cellular processes in diverse pathologies. The enzyme glutaminase (GLS) deaminates glutamine to form glutamate—the rate-limiting first step in glutamine metabolism. Using genetic and metabolic approaches, we demonstrate GLS and glutamine metabolism are required in SSCs to regulate osteoblast and adipocyte specification and bone formation. Mechanistically, transaminase-dependent α-ketoglutarate production is critical for the proliferation, specification, and differentiation of SSCs. Collectively, these data suggest stimulating GLS activity may provide a therapeutic approach to expand SSCs in aged individuals and enhance osteoblast differentiation and activity to increase bone mass.