The Sonic Hedgehog pathway plays important roles in mammalian inner ear development.Mutations of Shh, Smo and Gli3 lead to severe defects in mouse inner ear morphogenesis.However, knockout of Gli2 does not affect inner ear morphology or cochlear hair cell differentiation, suggesting that the Gli repressor function may be required for Hedgehog signaling during inner ear development.Suppressor of fused (Sufu) is a negative regulator of Hedgehog signaling and it functions to repress Gli activator and enhance Gli repressor activities.To understand the roles of Sufu and Gli transcription factors in mediating cochlear hair cell differentiation, we have analyzed the Pax2Cre,;Sufuflox/flox, Gli3P1-4/P1-4 and Gli3△699/△699 mutants using hair cell marker Myosin7a and supporting cell markers Sox2, P75 and Jag1.At E1 6.5, only one row of inner hair cells could be observed at the basal region of cochleae in the Pax2Cre;Sufuflox/flox mutants.Nevertheless, normal hair cells appeared at the medial region at E18.5, indicating that deletion of Sufu delays cochlear hair cell differentiation.In the Gli3P1-4/P1-4 mutant which lacked Gli3 repressor, cochlear hair cell differentiation was inhibited.Interestingly, in the Gli3△699/△699 mutant with excessive Gli3 repressor, hair cell differentiation was accelerated in the apical region of the cochlear duct.Our results suggest that Sufu and Gli3 repressor are essential factors that regulate the basal-to-apical progression of cochlear hair cell differentiation, supporting that Sonic Hedgehog signaling is required to control the dynamics of cochlear hair cell differentiation.