In amniotes, head movements are encoded by two types of vestibular hair cells (type I and type II) with unique morphology, physiology, and innervation. After hair cell destruction in mature rodents, supporting cells regenerate some type II hair cells, but no type I hair cells are replaced. The transcription factor Atoh1 is required for hair cell development, and Atoh1 is upregulated in supporting cells, the hair cell progenitors, in mature chickens and mice following hair cell damage. We investigated whether Atoh1 is required for type II hair cell regeneration in adult mice after genetic ablation of hair cells. First, we used a knock-in Atoh1 reporter to demonstrate that supporting cells in the utricle, a vestibular organ that detects linear acceleration of the head, upregulate Atoh1 expression by 7 days after hair cell destruction was initiated. Next, we labeled supporting cells prior to damage and fate-mapped them over time to test whether conditional deletion of Atoh1 from supporting cells prevented them from converting into hair cells after damage. In mice with normal Atoh1 expression, fate-mapped supporting cells in the adult utricle gave rise to hundreds of type II hair cells after hair cell destruction, but they did not form new type I hair cells. By contrast, mice with Atoh1 deletion prior to hair cell damage had only 10-20 fate-mapped type II hair cells per utricle at 3 weeks post-damage, and numbers did not change at 12 weeks after hair cell destruction. Supporting cells had normal cell shape and nuclear density up to 12 weeks after Atoh1 deletion. Similar observations were made in two other vestibular organs, the saccule and the lateral ampulla. Our findings demonstrate that Atoh1 is necessary in adult mouse supporting cells for regeneration of type II vestibular hair cells and that deletion of Atoh1 from supporting cells prior to damage does not appear to induce supporting cells to die or to proliferate. Copyright 2019 Elsevier B.V. All rights reserved.