Retinoic acid (RA) is one of the most important morphogens, and its embryonic distribution correlates with neural differentiation and positional specification in the developing central nervous system. To investigate the concentration-dependent effects of RA on neural differentiation of mouse embryonic stem cells (ES cells), we investigated the precise expression profiles of neural and regional specific genes by ES cells aggregated into embryoid bodies (EBs) exposed to various concentrations of RA or the BMP antagonist Noggin. RA promoted both neural differentiation and caudalization in a concentration-dependent manner, and the concentration of RA was found to regulate dorso-ventral identity, i.e., higher concentrations of RA induced a dorsal phenotype, and lower concentrations of RA induced a more ventral phenotype. The induction of the more ventral phenotype was due to the higher expression level of the N-terminus of sonic hedgehog protein (Shh-N) when treated with low concentration RA, as it was abrogated by an inhibitor of Shh signaling, cyclopamine. These findings suggest that the concentration of RA strictly and simultaneously regulates the neuralization and positional specification during differentiation of mouse ES cells and that it may be possible to use it to establish a strategy for controlling the identity of ES-cell-derived neural cells.