We developed a new end point of the mouse stem cell test (EST) for developmental neurotoxicity. We tested 2 developmental neurotoxicants, namely, lead (II) acetate and Aroclor 1254, using this EST. Our results showed that lead (II) acetate is nonembryotoxic, and Aroclor 1254 is weakly embryotoxic. To identify a new end point for developmental neurotoxicity, we used the default method of neuronal differentiation for D3 mouse embryonic stem cells with basic fibroblast growth factor (bFGF) and ascorbic acid. Flow cytometry and real-time polymerase chain reaction were used to quantify the inhibition of neuronal differentiation. Our results showed that both lead (II) acetate and Aroclor 1254 reduced the percentage of microtubule-associated protein 2 (MAP-2)-positive cells and the messenger RNA (mRNA) expression level of MAP-2 in a dose-dependent manner. These results suggested that these methods can be used to develop an additional end point of the EST for developmental neurotoxicity using default differentiation of mouse embryonic stem cells.