Objective: To investigate the role of the periotic mesenchyme (POM) in the development of sensory cells of developing auditory epithelium.Methods: Developing auditory epithelium with or without periotic mesenchyme was isolated from mice at embryonic days 11.5 (E11.5), E12.5 and E13.5, respectively, and cultured in vitro to an equivalent of E18.5's epithelium in vivo. Then, the explants were co-stained with antibodies targeting myosin VIIA, Sox2 and BrdU. Results: More hair cells in E11.5+7 DIV, E12.5+6 DIV and E13.5+5 DIV auditory epithelia were found upon culture with POM (225.90 ± 62.44, 476.94 ± 100.81, and 1386.60 ± 202.38, respectively) compared with the non-POM group (68.17 ± 23.74, 205.00 ± 44.23, and 1266.80 ± 38.84, respectively). Moreover, regardless of developmental stage, the mesenchymal tissue increased the amount of cochlear sensory cells as well as the ratio of differentiated hair cells to total sensory cells. Conclusions: The periotic mesenchyme promotes the development of cochlear sensory cells, and its ef-fect depends on the developmental stage of the auditory epithelium.