Experimental studies of inner ear development and regeneration, as well as investigations of the influences of sensory input on CNS development, often require a rapid and nearly complete elimination of the hair cells of the inner ear at any postnatal age. Although these cells can be killed by noise trauma or by exposure to ototoxic drugs, both of these interventions are highly variable in their efficacy, resulting in considerable differences in sensory functions among individual animals that receive the same treatment. Furthermore, much current research of the auditory and vestibular systems is conducted using mice, and the ears of mice are relatively resistant to the effects of many ototoxins. In response to these concerns and others, the Rubel and Palmiter labs at the University of Washington developed a transgenic mouse line (called Pou4f3DTR) in which the human form the diphtheria toxin receptor (also known as HB-EGF) is expressed under regulation of the Pou4f3 promoter. Because Pou4f3 is expressed by all hair cells (and relatively few other cells in the body), this mouse model permits the selective elimination of hair cells via 1–2 systemic injections of diphtheria toxin. This mouse line has been successfully used in studies of auditory CNS development and hair cell regeneration. This chapter provides an overview of this model, as well as detailed protocols for its use.