Precision of synaptic connections within neural circuits is essential for the accurate processing of sensory information. Specificity is exemplified at cellular and subcellular levels in the chick auditory brainstem, where nucleus magnocellularis (NM) neurons project bilaterally to nucleus laminaris (NL). Dorsal dendrites of NL neurons receive input from ipsilateral, but not contralateral, branches of NM axons whereas ventral dendrites are innervated by contralateral NM axons. This organization is analogous to that of the mammalian medial superior olive (MSO) and represents an important component of the circuitry underlying sound localization. However, the molecular mechanisms that establish segregated inputs to individual regions of NL neurons have not been identified. During synapse formation in NL, the EphA4 receptor is expressed in dorsal, but not ventral NL, neuropil, suggesting a potential role in targeting synapses to appropriate termination zones. Here, we directly tested this role by ectopically expressing EphA4 and disrupting EphA4 signaling using in ovo electroporation. We found that both misexpression of EphA4 and disruption of EphA4 signaling resulted in an increase in the number of NM axons that grow aberrantly across NL cell bodies into inappropriate regions of NL neuropil. EphA4 signaling is thus essential for targeting axons to distinct subsets of dendrites. Moreover, loss of EphA4 function resulted in morphological abnormalities of NL suggestive of errors in cell migration. These results suggest that EphA4 has multiple roles in the formation of auditory brainstem nuclei and their projections.