The midbrain-hindbrain boundary (MHB), also known as the isthmic organizer (IsO), plays a critical role in the developmental patterning of the posterior midbrain and anterior hindbrain. Understanding the wiring of this organizer's deeply conserved gene regulatory network is of significant interest for both evolutionary and neurodevelopmental biology. Various secreted signalling molecules and transcription factors have been identified as being important components for the formation and function of the MHB. Among these, FGF8 is considered a primary mediator of IsO activity; it directs anterior-posterior patterning and promotes the specification and maintenance of the MHB. While the core gene regulatory network governing MHB development is well-characterized, the direct interactions between key regulatory genes and the cis-regulatory elements that control their spatiotemporal expression remain poorly understood. This review summarizes the current knowledge of the gene regulatory network underlying the formation of the vertebrate midbrain-hindbrain boundary. We focus in particular on Fgf8 and its regulatory landscape from an evolutionary perspective.