In the last 2 decades, cell culture techniques for both mammalian embryonic stem cells and adult stem cells have developed and improved, and are now widely available. These stem cells are either pluri- or multi-potent, which makes them favorable for use in vitro developmental toxicity assays. Recent studies have reported several applications for embryonic and adult stem cells in cytotoxicity and developmental toxicity testing. These applications have the potential to provide alternative assessment techniques for evaluating toxic substances, and possibly reveal novel toxic and developmental effects that are difficult to investigate in humans because of ethical considerations. In this review, we describe some of the new approaches that use mammalian embryonic and adult stem cells in toxicological safety testing.