Dimethyl sulfoxide (DMSO) and ethanol are common organic solvents used for dissolving lipophilic substances for in vitro testing. However, DMSO is known to induce differentiation in embryonic stem (ES) and embryonic teratocarcinoma (EC) cells. In order to clarify if solvents like DMSO and ethanol have an influence on in vitro developmental toxicity test systems, the presented study has evaluated their effects on differentiation by using different test systems. ES and EC cells were transfected with a construct containing the mTert promoter combined with the green fluorescent protein gene (GFP). A down-regulation of mTert, a marker for undifferentiated cells, results in a lower expression of GFP, which could be measured by flow cytometry. Taking the specific characteristics of ES and EC cells into account this effect could be a hint for the interaction of DMSO with embryonic development. Additionally, the effects of the solvents ethanol and DMSO on Oct-4 expression, another marker for undifferentiated cells, were measured in wild-type ES cells. Both selected molecular markers demonstrated an induction of differentiation after exposure to DMSO; in wild-type ES cells at a concentration of 0.125% and in transgenic EC cells at a concentration of 0.25% DMSO. All other differences from controls, including those which attained a level of statistical significance, were minor or not dosage related in degree, or were not consistent over time and are, therefore, considered to be of little toxicological importance. In addition, a cytotoxicity test demonstrated that the solvents affected the employed molecular markers in non-cytotoxic concentrations. The ES cells were the most sensitive towards the cytotoxic effects of the solvent DMSO while the EC cells were more sensitive when treated with the solvent ethanol.