The homeodomain transcription factor vHNF1 plays an essential role in the patterning of the caudal segmented hindbrain, where it participates in the definition of the boundary between rhombomeres (r) 4 and 5 and in the specification of the identity of r5 and r6. Understanding the molecular basis of vHnf1 own expression therefore constitutes an important issue to decipher the regulatory network governing hindbrain patterning. We have identified a highly conserved 800-bp enhancer element located in the fourth intron of vHnf1 and whose activity recapitulates vHnf1 neural expression in transgenic mice. Functional analysis of this enhancer revealed that it contains two types of essential motifs, a retinoic acid response element and two half T-MARE sites, indicating that it integrates direct inputs from the retinoic acid signaling cascade and MAF-related factors. Our data suggest that MAFB, which is itself regulated by vHNF1, acts as a positive modulator of vHnf1 in r5 and r6, whereas another MAF-related factor is absolutely required for the expression of vHnf1 in both the hindbrain and the spinal cord. We propose a model accounting for the initiation and maintenance phases of vHnf1 expression and for the establishment of the r4/r5 boundary, based on cooperative contributions of Maf factors and retinoic acid signaling.