Ethanol up to 0.5% is recommended for use as a solvent in mouse embryonic stem cell (mESC) differentiation assays such as the Embryonic Stem Cell Test. However, lower concentrations have been shown by some to alter mESC differentiation. In this study, we further explored the dose‐ and time‐dependent affects of ethanol on mESC differentiation into cardiomyocytes using the hanging drop culture method and J1 mESC. Among control cultures (no ethanol), rhythmically contracting cells were observed in cultures after 11 days at a frequency of 蠅64%. With continuous ethanol exposure, a significant (p 蠄 0.05) decrease in the fraction of cultures containing contracting cells was observed by 蠅68% at ethanol concentrations of 0.3% and higher and was accompanied by a 蠅30% loss of cells. With ethanol exposure during the first two days of culture only (the embryoid body stage), no significant change in the frequency of contracting cells per culture, or number of cells, was observed after 11 days at ethanol concentrations up to 0.5%. These results suggest that ethanol concentrations below 0.5% can reduce the frequency of contracting cells, at least in part, by reducing the number of cells during differentiation, and that the most sensitive time‐frame for ethanol toxicity is after the embryoid body stage. Grant Funding Source: Supported by NSF DUE‐0622421