Establishment of in vitro to insulin and transferrin-se-fibronectin (ITSFn) training method based on embryonic stem cells neural developmental toxicity evaluation model, and its effectiveness is verified, the refinement of neural development toxicity evaluation system. Methods embryonic stem cells suspended drops, suspension culture, insulin, transferrin, adding selenium, fibronectin induces neuronal differentiation, in combination with cytotoxic MTT law to build the evaluation model of neurodevelopmental toxicity in vitro, and detection of different concentrations of penicillin G, phenytoin and 5-fluorouracil effect under the condition of embryonic stem cell neural differentiation, ability, judgment the neurodevelopmental toxicity. Resultspenicillin G, phenytoin and 5-fluorouracil ID50D3nestin were 0.017μ g/ml, 49.40μ g/ml, 1139 μ g/ml, ID50D3EN-1respectively 0.031μ g/ml, 25.63μ g/ml, 1762μ g/ ml, its developmental toxicity determination in turn were no, weak and strong. ConclusionsBased on ITSFn neural development toxicity in vitro models to evaluate the correct judgement of 5-fluorouracil, phenytoin and penicillin G developmental toxicity, can be used for in vitro neural developmental toxicity screening and evaluation.