In the avian auditory system, the neural network for computing the localization of sound in space begins with bilateral innervation of nucleus laminaris (NL) by nucleus magnocellularis (NM) neurons. We used antibodies against the neural specific markers Hu C/D, neurofilament, and SV2 together with retrograde fluorescent dextran labeling from the contralateral hindbrain to identify NM neurons within the anlage and follow their development. NM neurons could be identified by retrograde labeling as early as embryonic day (E) 6. While the auditory anlage organized itself into NM and NL in a rostral-to-caudal fashion between E6 and E8, labeled NM neurons were visible throughout the extent of the anlage at E6. By observing the pattern of neuronal rearrangements together with the pattern of contralaterally projecting NM fibers, we could identify NL in the ventral anlage. Ipsilateral NM fibers contacted the developing NL at E8, well after NM collaterals had projected contralaterally. Furthermore, the formation of ipsilateral connections between NM and NL neurons appeared to coincide with the arrival of VIIIth nerve fibers in NM. By E10, immunoreactivity for SV2 was heavily concentrated in the dorsal and ventral neuropils of NL. Thus, extensive pathfinding and morphological rearrangement of central auditory nuclei occurs well before the arrival of cochlear afferents. Our results suggest that NM neurons may play a central role in formation of tonotopic connections in the auditory system.