A basic helix-loop-helix transcriptional factor, NeuroD2, plays important roles in neuronal differentiation and survival. We introduced the tetracycline-dependent NeuroD2 expression system to embryonic stem (ES) cells and studied the role of NeuroD2 in the neuronal differentiation. The addition of doxycycline induced the expression of NeuroD2 after 24 h and the differentiation to neurons after 3 days in ES cells, which are transfected with vectors composed of reverse tetracycline-controlled transactivator with cytomegarovirus promoter and NeuroD2 with tetracycline response element. Treatment with doxycycline for 3 days induced neuronal differentiation, but not within 1 day; furthermore NeuroD2 was detected in the nucleus 3 days after treatment, but also not within 1 day. The results suggest that the expression of NeuroD2 requires an appropriate period of about 3 days to elicit neuronal differentiation in ES cells.