The Msx and Dlx families of homeobox proteins are important regulators for embryogenesis. Loss of Msx1 in mice results in multiple developmental defects including craniofacial malformations. Although Dlx5 is widely expressed during embryonic development, targeted null mutation of Dlx5 mainly affects the development of craniofacial bones. Msx1 and Dlx5 show overlapping expression patterns during frontal bone development. To investigate the functional significance of Msx1/Dlx5 interaction in regulating frontal bone development, we generated Msx1 and Dlx5 double null mutant mice. In Msx1−/−;Dlx5−/− mice, the frontal bones defect was more severe than that of either Msx1−/− or Dlx5−/− mice. This aggravated frontal bone defect suggests that Msx1 and Dlx5 function synergistically to regulate osteogenesis. This synergistic effect of Msx1 and Dlx5 on the frontal bone represents a tissue specific mode of interaction of the Msx and Dlx genes. Furthermore, Dlx5 requires Msx1 for its expression in the context of frontal bone development. Our study shows that Msx1/Dlx5 interaction is crucial for osteogenic induction during frontal bone development. genesis 48:645–655, 2010. © 2010 Wiley-Liss, Inc.