The homeobox-containing gene variant hepatocyte nuclear factor-1 (vHnf1) has recently been shown to be involved in zebrafish caudal hindbrain specification, notably in the activation of MafB and Krox20 expression. We have explored this regulatory network in the chick by in ovo electroporation in the neural tube. We show that misexpression of vHnf1 confers caudal identity to more anterior regions of the hindbrain. Ectopic expression of mvHnf1 leads to ectopic activation of MafB and Krox20, and downregulation of Hoxb1 in rhombomere 4. Unexpectedly, mvhnf1 strongly upregulates Fgf3 expression throughout the hindbrain, in both a cell-autonomous and a non-cell-autonomous manner. Blockade of FGF signaling correlates with a selective loss of MafB and Krox20 expression, without affecting the expression of vHnf1, Fgf3, or Hoxb1. Based on these observations, we propose that in chick, as in zebrafish, vHnf1 acts with FGF to promote caudal hindbrain identity by activating MafB and Krox20 expression. However, our data suggest differences in the vHnf1 downstream cascade in different vertebrates. Developmental Dynamics 234:567–576, 2005. © 2005 Wiley-Liss, Inc.