The application of mesenchymal stem cells (MSCs) for myocardial repair following ischemic injury is of strong interest, but current knowledge regarding the survival and retention of differentiation potency of stem cells under ischemic conditions is limited. The present study investigated the effects of ischemia and its components (hypoxia and glucose depletion) on MSC viability and multipotency. We demonstrate that MSCs have a profoundly greater capacity to survive under conditions of ischemia compared with cardiomyocytes, measured by detecting changes in cellular morphology, caspase activity and phosphatidylserine exposure. MSCs were also resistant to exposure to hypoxia (0.5% O(2)), as well as inhibition of mitochondrial respiration with 2,4-dinitrophenol for 72 hours, indicating that in the absence of oxygen, MSCs can survive using anaerobic ATP production. Glucose deprivation (glucose-free medium in combination with 2-deoxyglucose) induced rapid death of MSCs. Depletion of cellular ATP occurred at a lower rate during glucose deprivation than during ischemia, suggesting that glycolysis has specific prosurvival functions, independent of energy production in MSCs. After exposure to hypoxic or ischemic conditions, MSCs retained the ability to differentiate into chondrocytes and adipocytes and, more importantly, retained cardiomyogenic potency. These results suggest that MSCs are characterized by metabolic flexibility, which enables them to survive under conditions of ischemic stress and retain their multipotent phenotype. These results highlight the potential utility of MSCs in the treatment of ischemic disease.