Cleft palate is a common major birth defect resulting from disruption of palatal shelf growth, elevation, or fusion during fetal palatogenesis. Whereas the molecular mechanism controlling palatal shelf elevation is not well understood, a prevailing hypothesis is that region-specific accumulation of hyaluronan, a predominant extracellular glycosaminoglycan in developing palatal mesenchyme, plays a major role in palatal shelf elevation. However, direct genetic evidence for a requirement of hyaluronan in palate development is still lacking. In this study, we show that Has2, 1 of 3 hyaluronan synthases in mammals, plays a major role in hyaluronan synthesis in the neural crest-derived craniofacial mesenchyme during palatogenesis in mice. We analyzed developmental defects caused by tissue-specific inactivation of throughout the cranial neural crest lineage or specifically in developing palatal or mandibular mesenchyme, respectively, using , and transgenic mice. Inactivation of either throughout the neural crest lineage or specifically in the developing palatal mesenchyme caused reduced palatal shelf size and increased palatal mesenchyme cell density prior to the time of normal palatal shelf elevation. Whereas both ; and ; mutant mice exhibit cleft palate at complete penetrance, the ; fetuses showed dramatically reduced mandible size and complete failure of palatal shelf elevation, whereas ; fetuses had normal mandibles and delayed palatal shelf elevation. All ; pups showed reduced mandible size and about 50% of them had cleft palate with disruption of palatal shelf elevation. Results from explant culture assays indicate that disruption of palatal shelf elevation in ; mutant fetuses resulted from physical obstruction by the malformed mandible and tongue. Together, these data indicate that hyaluronan plays a crucial intrinsic role in palatal shelf expansion and timely reorientation to the horizontal position above the tongue as well as an important role in mandibular morphogenesis that secondarily affects palatal shelf elevation.