The Notch signaling pathway has a crucial role in the differentiation of hair cells and supporting cells by mediating “lateral inhibition” via the ligands Delta-like1 (Dll1) and Jagged2 (Jag2) and the effectors Hes1 and Hes5 during mammalian inner ear development. Recently, another Notch ligand, Jagged1 (Jag1)-dependent Notch activation, has been revealed to be important for the determination of the prosensory region in the earlier stage before cell differentiation. However, little is known about the effectors of the Notch pathway in this context. P27Kip1, a cyclin-dependent kinase inhibitor, is also known to demarcate the prosensory region in the cochlear primordium, which consists of the sensory progenitors that have completed their terminal mitoses. Hes1 reportedly promotes precursor cell proliferation through the transcriptional down-regulation of p27Kip1 in the thymus, liver, and brain. In this study, we observed Hes1 as a mediator between the Notch signaling pathway and the regulation of proliferation of sensory precursor cells by p27Kip1 in the developing cochlea. We showed that Hes1, but not Hes5, was weakly expressed at the time of onset of p27Kip1. The expression pattern of Hes1 prior to cell differentiation was similar to that of activated Notch1. P27Kip1 was up-regulated and BrdU-positive S-phase cells were reduced in the developing cochlear epithelium of Hes1 null mice. These results suggest that the Notch-Hes1 pathway may contribute to the adequate proliferation of sensory precursor cells via the potential transcriptional down-regulation of p27Kip1 expression and play a pivotal role in the correct prosensory determination. © 2009 Wiley-Liss, Inc.