Embryonic cells before implantation are exposed to a hypoxic condition and dependent on anaerobic metabolism. Human embryonic stem cells (HESCs) derived from pre-implantation blastocyst also grow well in hypoxic conditions. Expecting that the differentiating HESCs might mimic anaerobic-to-aerobic metabolic transition of the early human life, we examined the mitochondria-related changes in these cells. We observed that mitochondrial mass and mitochondrial DNA content were increased with differentiation, which was accompanied by the increase of the amount of ATP (4-fold) and its by-product reactive oxygen species (2.5-fold). The expression of various antioxidant enzymes including mitochondrial and cytoplasmic superoxide dismutases, catalase, and peroxiredoxins showed a dramatic change during the early differentiation. In conclusion, HESC differentiation was followed by dynamic changes in mitochondrial mass, ATP and ROS production, and antioxidant enzyme expressions. Therefore, the HESCs would serve as a good model to examine the mitochondrial biology during the early human differentiation.