In order to prevent birth defects, toxicology programs have been designed to identify toxicities that may potentially be encountered in human embryos. With appropriate toxicity data sets, acceptable exposure levels and actual safety of prescription and nonprescription drugs as well as environmental chemicals could be established for individuals that are more vulnerable to chemical exposure, such as pregnant women and their unborn children. The gathering of such embryotoxicity data is currently performed in animal models. To reduce the spending of live animals, an assortment of in vitro assays has been proposed.In this chapter, the embryonic stem cell test (EST) is reviewed as an alternative model for testing embryotoxicity. In contrast to most in vitro toxicity assays, the EST uses two permanent cell lines: murine 3T3 fibroblasts and murine embryonic stem cells (ESCs). To establish developmental toxicity, the difference in sensitivity towards the cytotoxic potential of a given test compound between the adult and the embryonic cells is compared with an MTT assay. In addition, the EST contrasts the inhibition of development that a test compound may cause utilizing the in vitro differentiation potential of the ESCs.We describe here protocols to culture both cell lines as well as the differentiation of the ESCs into cardiomyocytes. Classically, the EST assesses developmental toxicity through counting of contracting cardiomyocyte agglomerates, which will be described as one endpoint. Although this classic EST has been validated in an EU-wide study, tremendous problems exist with the choice of endpoints, the EST's predictivity, and the associated costs. We therefore also give details on the more recently introduced molecular analysis of cardiomyocyte-specific mRNAs, which already has been used to successfully predict developmental toxicity. Moreover, this chapter will explain a method to evaluate developmental bone toxicity and hencewith an experimental setup to differentiate ESCs into osteoblasts is presented along with two endpoint analyses that will establish generation of osteoblasts as well as their calcification in culture. The various differentiation endpoints may be set into relation to the cytotoxicity that the same test compound causes to ultimately predict the potential of a compound to excite developmental toxicity in vivo.