Tbx1 belongs to the family of T-box containing transcription factors. In humans, TBX1 is implicated in the etiology of the DiGeorge syndrome. Inactivation of the Tbx1 gene in mice produces a variety of malformations including abnormal branching of the heart outflow tract, deficiencies in the branchial arch derivatives, agenesis of pharyngeal glands and abnormal development of the auditory system. We analyze here the middle and inner ear phenotypes of the Tbx1 null mice. The middle ear is strongly affected. Its skeletal components are malformed to varying degrees, some being slightly hypoplastic and others completely absent. However, a seemingly normal-looking tympanic membrane can still be recognized. Middle ear anomalies are associated with other skeletal deficiencies in the branchial arch-derived skeleton. These phenotypes derive from a combination of the failure of the posterior branchial arches to develop and the misrouting of neural crest cells. The inner ears of Tbx1(-/-) animals are hypoplastic. No vestibular or cochlear structures are detectable, but the endolymphatic duct, the cochleovestibular ganglia and residual sensory patches are still identifiable. Molecular analyses revealed a seemingly normal spatial distribution of a variety of patterning markers in the otic vesicles of Tbx1 null mutants at E9.0. However, 1 day later, several of these markers presented altered domains of expression in the otocysts of these mutant embryos, suggesting that Tbx1 is not required for the establishment of spatial patterns in the otocyst, but rather for their maintenance. The inability of the Tbx1(-/-) embryos to keep properly segregated functional domains in the otocyst is likely the cause of the strong inner ear phenotypes observed in these mutants.