Hox proteins are well known for executing highly specific functions in vivo, but our understanding of the molecular mechanisms underlying gene regulation by these fascinating proteins has lagged behind. The premise of this review is that an understanding of gene regulation-by any transcription factor-requires the dissection of the cis-regulatory elements that they act upon. With this goal in mind, we review the concepts and ideas regarding gene regulation by Hox proteins and apply them to a curated list of directly regulated Hox cis-regulatory elements that have been validated in the literature. Our analysis of the Hox-binding sites within these elements suggests several emerging generalizations. We distinguish between Hox cofactors, proteins that bind DNA cooperatively with Hox proteins and thereby help with DNA-binding site selection, and Hox collaborators, proteins that bind in parallel to Hox-targeted cis-regulatory elements and dictate the sign and strength of gene regulation. Finally, we summarize insights that come from examining five X-ray crystal structures of Hox-cofactor-DNA complexes. Together, these analyses reveal an enormous amount of flexibility into how Hox proteins function to regulate gene expression, perhaps providing an explanation for why these factors have been central players in the evolution of morphological diversity in the animal kingdom.