In these studies, we explored for the first time the molecular relationship between the paired-domain-containing transcription factor,Pax9, and the ectodysplasin (Eda) signaling pathway during mouse incisor formation. Mice that were deficient in bothPax9andEdawere generated, and the status of dentition analyzed in all progeny using gross evaluation and histomorphometric means. When compared to wildtype controls,Pax9+/–Eda–/–mice lack mandibular incisors. Interestingly,FgfandShhsignaling are down-regulated whileBmp4andLef1appear unaffected. These findings suggest thatPax9-dependent signaling involves theEdapathway and that this genetic relationship is important for mandibular incisor development. Studies of records of humans affected by mutations inPAX9lead to the congenital absence of posterior dentition but interestingly involve agenesis of mandibular central incisors. The latter phenotype is exhibited by individuals withEDAorEDARmutations. Thus, it is likely thatPAX9, in addition to playing a role in the formation of more complex dentition, is also involved withEDAsignaling in the initiation of odontogenesis within the incisal domain.