The epidermis is a derivative of the surface ectoderm. It forms a protective barrier and specific appendages including hair, nails, and different eccrine glands. The surface ectoderm also forms the epithelium of the oral cavity and tongue, which develop a slightly different barrier and form different appendages such as teeth, filiform papillae, taste papillae, and salivary glands. How this region-specific differentiation is genetically controlled is largely unknown. We show here that Pax9, which is expressed in the epithelium of the tongue but not in skin, regulates several aspects of tongue-specific epithelial differentiation. In Pax9-deficient mice filiform papillae lack the anterior-posterior polarity, a defect that is associated with temporal-spatial changes in Hoxc13 expression. Barrier formation is disturbed in the mutant tongue and genome-wide expression profiling revealed that the expression of specific keratins (Krt), keratin-associated proteins, and members of the epidermal differentiation complex is significantly down-regulated. In situ hybridization demonstrated that several 'hard' keratins, Krt1-5, Krt1-24, and Krt2-16, are not expressed in the absence of Pax9. Notably, specific 'soft' keratins, Krt2-1 and Krt2-17, normally weakly expressed in the tongue but present at high levels in skin and in orthokeratinized oral dysplasia are up-regulated in the mutant tongue epithelium. This result indicates a partial trans-differentiation to an epithelium with skin-specific characteristics. Together, our findings show that Pax9 regulates appendage formation in the mammalian tongue and identify Pax9 as an important factor for the region-specific differentiation of the surface ectoderm.