In mammalian embryos, transient Fgf8 expression defines the developing isthmic region, lying between the midbrain and the first rhombomere, but there has been uncertainty about the existence of a distinct isthmic segment in postnatal mammals. Retinoic acid (RA) directly represses Fgf8 through a RARE-mediated mechanism that promotes repressive chromatin, thus providing valuable insight into the mechanism of RA-FGF antagonism during progenitor cell differentiation. Fgf8 encodes a key signaling factor, and its precise regulation is essential for embryo patterning.