The monolayer culture of embryonic stem (ES) cells and embryoid body (EB) formation were carried out under various oxygen tensions (i.e., 5% O-2, 20% O-2, and 40% O-2). The ES cells cultured in 5% 02 and 20% O-2 exhibited decreased specific alkaline phosphatase (AP) activity with culture time, whereas the ES cells cultured in 40% O-2 retained their specific AP activity until culture day 3. The ES cells in 40% O-2 maintained an octamer-binding transcription factor 4 (Oct-4; a marker of pluripotent undifferentiated ES cells) gene expression level higher than that at other oxygen tensions. The EB formed in 40% 02 maintained an Oct-4 gene expression level higher than that at other oxygen tensions. The generation of cardiomyocytes and the decline in Oct-4 gene expression level were earliest in the EB formed in 20% O-2. It was suggested that the culture condition under a high oxygen tension (40% 02) retards the differentiation of ES cells and a normal oxygen tension (20% 02) allows spontaneous cell differentiation.