A flat epithelium (FE) may be found in the vestibular end organs of humans and mice with vestibular dysfunction. However, the pathogenesis of FE is unclear and inducing hair cell (HC) regeneration is challenging, as both HCs and supporting cells (SCs) in vestibular FE are damaged. To determine the cellular origin of vestibular FE and examine its response to Atoh1 overexpression, we fate-mapped vestibular epithelial cells in three transgenic mouse lines (vGlut3-iCreER(T2):Rosa26(tdTomato), GLAST-CreER-(T2):Rosa26(tdTomato) and Plp-CreER(T2):Rosa26(tdTomato)) after inducing a lesion by administering a high dose of streptomycin. Atoh1 overexpression in vestibular FE was mediated by an adeno-associated virus serotype 8 (AAV8) vector. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, was administered with AAV8 to enhance Atoh1 overexpression. The transduction efficiency and population of myosin Vila-positive cells were analyzed. A small number of HCs were present in vestibular FE. FE did not show broad GLAST-Cre or Plp-Cre expression, unlike the original SCs. SAHA dramatically enhanced AAV8-mediated exogenous gene overexpression, and Atoh1 overexpression plus SAHA promoted myosin Vila expression in FE cells. Our data provide insight into FE formation and will facilitate studies of gene therapy for vestibular FE. (C) 2020 Elsevier B.V. All rights reserved.