Many epidemiological and animal exposure studies have suggested that exposure to environmental substances is a major risk factor for developmental neurotoxicity (DNT), such as in autism, and is related to the increasing relevance of neurodevelopmental disorders. Recent efforts have led to the development of various in vitro approaches that use cell lines and pluripotent stem cells to assess numerous environmental substances. In this study, we developed a method for assessing DNT using a mouse embryonic stem (mES) cell model that focuses on differentiation into neuronal cells (neural cells and astrocytes). Using this model system, we found that six insecticides inhibited the differentiation of neural precursor cells into astrocytes. Our data indicated that the effects of insecticides on glial differentiation were more sensitive than those of several DNT markers reported in previous studies. This mES cell model can make a quick assessment of DNT potential and may be a useful tool for screening substances with potential to induce DNT.