The Hox genes are best known as regulators of anterior-posterior identity along the embryonic axis, including in the central nervous system. Here (p. 1492), Alex Gould and co-workers identify a function for Hox proteins in regulating cell segregation and boundary formation between rhombomeres of the mammalian and chick hindbrain. Hox4 family members show an anterior expression border at the r6/7 boundary, and Hox4 depletion inhibits formation of this boundary. Conversely, cells ectopically expressing Hox4 cluster together and display boundary-like features at their borders. These data suggest that the presence of an interface between Hox4-expressing and non-expressing cells is required for cell segregation at the r6/r7 boundary. Mechanistically, the authors show that Eph/ephrin genes known to be involved in boundary formation are regulated by Hox4, and Hox4 appears to control cell shape in a non-autonomous fashion - promoting apical enlargement on either side of the rhombomere interface. Other Hox proteins appear to have similar activities, suggesting a conserved function for this protein family in regulating cell segregation.