An important feature of stem cells is their maintenance in their respective hypoxic niche. Survival in this low-oxygen microenvironment requires significant metabolic adaptation. We demonstrated that mouse HSCs utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands. We have adapted various tools for characterization of the metabolic properties of hematopoietic stem cells (HSCs). These techniques include flow cytometric profiling of HSCs based on mitochondrial potential and NADH fluorescence as well as measurement of ATP content, oxygen consumption rate, and glycolytic flux in purified HSCs.