Hox genes are known key regulators of embryonic segmental identity, but little is known about the mechanisms of their action. To address this issue, we have analyzed how Hoxa2 specifies segmental identity in the second branchial arch. Using a subtraction approach, we found that Ptx1 was upregulated in the second arch mesenchyme of Hoxa2 mutants. This upregulation has functional significance because, in Hoxa2(-/-);Ptx1(-/-) embryos, the Hoxa2(-/-) phenotype is partially reversed. Hoxa2 interferes with the Ptx1 activating process, which is dependent on Fgf signals from the epithelium. Consistently, Lhx6, another target of Fgf8 signaling, is also upregulated in the Hoxa2(-/-) second arch mesenchyme. Our findings have important implications for the understanding of developmental processes in the branchial area and suggest a novel mechanism for mesenchymal patterning by Hox genes that acts to define the competence of mesenchymal cells to respond to skeletogenic signals.