Application of ouabain to the round window membrane of the gerbil selectively induces the death of most spiral ganglion neurons and thus provides an excellent model for investigating the survival and differentiation of embryonic stem cells (ESCs) introduced into the inner ear. In this study, mouse ESCs were pretreated with a neural-induction protocol and transplanted into Rosenthal's canal (RC), perilymph, or endolymph of Mongolian gerbils either 1-3 days (early post-injury transplant group) or 7 days or longer (late post-injury transplant group) after ouabain injury. Overall, ESC survival in RC and perilymphatic spaces was significantly greater in the early post-injury microenvironment as compared to the later post-injury condition. Viable clusters of ESCs within RC and perilymphatic spaces appeared to be associated with neovascularization in the early post-injury group. A small number of ESCs transplanted within RC stained for mature neuronal or glial cell markers. ESCs introduced into perilymph survived in several locations, but most differentiated into glia-like cells. ESCs transplanted into endolymph survived poorly if at all. These experiments demonstrate that there is an optimal time window for engraftment and survival of ESCs that occurs in the early post-injury period.