Introduction: During normal mammalian development, most of the cells within a developing embryo exist in an environment with an oxygen partial pressure (pO₂) that is less than 40 mmHg. Most in vitro culture of embryonic stem (ES) cells and their differentiated progeny is done in a humidified 5% CO₂ / 95% air incubator that produces a gas phase pO₂ (pO_2gas) of 142 mmHg. As an initial step to test whether control of cellular pO₂ (pO_2cell) to more physiological levels significantly affects ES cell differentiation, we studied the cardiomyocytic differentiation of murine ES cells at four different controlled pO_2cell levels. Methods: Embryoid bodies (EBs) were formed in hanging drops containing 200 ES cells in 20 ul of DMEM supplemented with 10% FBS. After culture for 3 - 4 days in hanging drops, EBs were transferred into dishes that had fibronectin-coated silicone rubber membrane bottoms, to which the cells attached and grew for an additional 6 – 10 days in a serum-free ITS medium. The pO_2gas was controlled by placing culture dishes in small airtight containers purged with premixed gas containing 5% CO₂ and either 0, 1, or 5% oxygen, resulting in environments with pO_2gas values of 0, 7, or 36 mmHg, respectively. Culture in a standard incubator (142 mmHg) was used as a control. By using silicone rubber membrane-based dishes for all conditions, the pO_2cell was nearly identical to pO_2gas. Cardiomyocytic differentiation was assessed by quantifying the surface area containing spontaneously contracting cells and by flow cytometric analysis of dispersed cells immunostained with an antibody to sarcomeric myosin heavy chain (MF-20). Four experiments were conducted using the protocol described above, 3 with the J1 cell line, and 1 with the R1 cell line. Results: Between 0.4 and 1% (average 0.7%) of cells were positive for MF-20 after being cultured as described for 10 days at a pO_2cell of 142 mmHg. Decreasing the pO_2cell to 7 mmHg caused the percentage of MF-20 positive cells to increase to between 3 and 6% (average 5.2%). On average there was an 8-fold increase in the fraction of cells that were MF-20 positive at a pO_2cell of 7 mmHg relative to 142 mmHg, and this difference was highly significant (p<0.005). In the two experiments that included pO_2cell conditions of 36 and 0 mmHg, the average MF-20 positive cell fraction was 4 and 2.5%, respectively. The percentage of the dish surface covered with contracting cells correlated well to the MF-20 results. Other experiments with the CCE and D3 cell lines also qualitatively showed similar increases in cardiomyocyte yield from low oxygen culture. In a single initial experiment, the cardiomyocytic cell fraction after 10 days in culture increased to 20% of cells if reduced pO_2cell conditions (7 mmHg) were combined with the addition of 0.2 mM ascorbic acid to the culture medium, an amplification of more than 20-fold compared to control cultures maintained with ascorbic acid at a pO_2cell of 142 mmHg. Conclusions: Culture at controlled pO_2cell conditions significantly below 142 mmHg promotes differentiation of ES cells into cardiomyocytes. Work is ongoing to determine the optimal pO₂ condition for each of the cell lines, and to further study whether reduced oxygen culture can be used synergistically with other cardiac promoting molecules and growth factors to further enhance the yield of cardiomyocytes obtained from the differentiation of ES cells.