The retinoblastoma protein (pRb) is required for cell-cycle exit of embryonic mammalian hair cells but is not required for hair cell fate determination and early differentiation, and this provides a strategy for hair cell regeneration by manipulating the pRb pathway. To reveal the mechanism of pRb functional modification in the inner ear, we compared the effects of attenuated pRb phosphorylation by an inhibitor of the Mitogen-Activated Protein (MAP) kinase pathway and an inhibitor of the Rb-Raf-1 interaction on cultured chicken otocysts. We demonstrated that the activity of pRb is correlated with its phosphorylation state, which is regulated by a newly established cell cycle-independent pathway mediated by the physical interaction between Raf-1 and pRb. The phosphorylation of pRb plays an important role during the early stage of inner ear development, and attenuated phosphorylation in progenitor cells leads to cell cycle arrest and increased apoptosis along with a global down-regulation of the genes involved in cell cycle progression. Our study provides novel routes to modulate pRb function for hair cell regeneration.