The vertebrate Dlx genes, generally organized as tail-to-tail bigene clusters, are expressed in the branchial arch epithelium and mesenchyme with nested proximodistal expression implicating a code that underlies the fates of jaws. Little is known of the regulatory architecture that is responsible for Dlx gene expression in developing arches. We have identified two distinct cis-acting regulatory sequences, I12a and I56i, in the intergenic regions of the Dlx1/2 and Dlx5/6 clusters that act as enhancers in the arch mesenchyme. LacZ transgene expression containing I12a is restricted to a subset of Dlx-expressing ectomesenchyme in the first arch. The I56i enhancer is active in a broader domain in the first arch mesenchyme. Expression of transgenes containing either the I12a or the I56i enhancers is dependent on the presence of epithelium between the onset of their expression at E9-10 until independence at E11. Both enhancers positively respond to FGF8 and FGF9; however, the responses of the reporter transgenes were limited to their normal domain of expression. BMP4 had a negative effect on expression of both transgenes and counteracted the effects of FGF8. Furthermore, bosentan, a pharmacological inhibitor of Endothelin-1 signaling caused a loss of I56i-lacZ expression in the most distal aspects of the expression domain, corresponding to the area of Dlx-6 expression previously shown to be under the control of Endothelin-1. Thus, the combinatorial branchial arch expression of Dlx genes is achieved through interactions between signaling pathways and intrinsic cellular factors. I56i drives the entire expression of Dlx5/6 in the first arch and contains necessary sequences for regulation by at least three separate pathways, whereas I12a only replicates a small domain of endogenous expression, regulated in part by BMP-4 and FGF-8.