Mitochondria are the powerhouse of the cell, providing energy in the form of ATP and NADH for cellular activities. In the pluripotent state, mitochondrial activity is relatively limited, but mitochondrial energy production increases as a cell differentiates and typically moves from a hypoxic to normoxic environment. Initial studies showed that a small molecule probe that interfered with mitochondrial function selective killed pluripotent cells but not the differentiated lineages. With the goal of stem cell therapeutics, transplanting stem cells to patients to correct a disease could be potentially dangerous if a population of stem cells that fail to differentiate moved to an environment that could induce teratomas. The probe was tested to determine if it could be useful for characterizing the differentiation process, with the goal of making stem cell therapies safer. Mechanistic studies were also completed to understand important aspects of the mitochondrial function in stem cell pluripotency and differentiation pathways. A novel signaling pathway was identified that is required for activating mitochondrial function as the stem cells differentiate and move from a hypoxic to normoxic environment. Future studies to further these outcomes will focus on optimizing the probe for stem cell differentiation studies and identifying key steps in the signaling pathway that are required to induce mitochondrial function during differentiation.