During embryonic development, the anterior-posterior body axis is specified in part by the combinatorial activities of Hox genes. Given the poor DNA binding specificity of Hox proteins, their interaction with cofactors to regulate target genes is critical. However, few regulatory partners or downstream target genes have been identified. Herein, we demonstrate that Hox11 paralogous proteins form a complex with Pax2 and Eya1 to directly activate expression of Six2 and Gdnf in the metanephric mesenchyme. We have identified the binding site within the Six2 enhancer necessary for Hox11-Eya1-Pax2-mediated activation and demonstrate that this site is essential for Six2 expression in vivo. Furthermore, genetic interactions between Hox11 and Eya1 are consistent with their participation in the same pathway. Thus, anterior-posterior-patterning Hox proteins interact with Pax2 and Eya1, factors important for nephrogenic mesoderm specification, to directly regulate the activation of downstream target genes during early kidney development.