The morphogenesis of the vertebrate hindbrain involves the generation of metameric units called rhombomeres (r), and Krox20 encodes a transcription factor that is expressed in r3 and r5 and plays a major role in this segmentation process. Our knowledge of the basis of Krox20 regulation in r3 is rather confusing, especially concerning the involvement of Hox factors. To investigate this issue, we studied one of the Krox20 hindbrain cis-regulatory sequences, element C, which is active in r3-r5 and which is the only initiator element in r3. We show that element C contains multiple binding sites for Meis and Hox/Pbx factors and that these proteins synergize to activate the enhancer. Mutation of these binding sites allowed us to establish that Krox20 is under the direct transcriptional control of both Meis (presumably Meis2) and Hox/Pbx factors in r3. Furthermore, our data indicate that element C functions according to multiple modes, in Meis-independent or -dependent manners and with different Hox proteins, in r3 and r5. Finally, we show that the Hoxb1 and Krox20 expression domains transiently overlap in prospective r3, and that Hoxb1 binds to element C in vivo, supporting a cell-autonomous involvement of Hox paralogous group 1 proteins in Krox20 regulation. Altogether, our data clarify the molecular mechanisms of an essential step in hindbrain patterning. We propose a model for the complex regulation of Krox20, involving a novel mode of initiation, positive and negative controls by Hox proteins, and multiple direct and indirect autoregulatory loops.