The anatomy of the cranial peripheral nervous system has been studied for over a century, yet surprisingly little is known about how the nerves are guided to their targets. The study of the development of these nerves has important implications for our understanding of craniofacial anomalies and possible treatments for both injury and genetic disorders of nerve development such as Goldenhar-Gorlin syndrome. We have discovered that retinoic acid (RA) may play a role in the development of the trigeminal nerve. Inhibition of retinoic acid receptors (RAR) results in trigeminal nerves that become unbundled or defasciculated in the eye region. To further understand how RA is affecting trigeminal development we searched for genes downregulated in response to RAR inhibition by the inhibitor BMS-453 and have identified neurotrophin-3 (NT-3), activated leukocyte cell adhesion molecule (ALCAM) and Semaphorin 4B (Sema4B). We have analyzed the expression patterns of Sema4B and NT-3 by in situ hybridization and have found NT-3 expression in the eye and Sema4B in the embryonic target of the trigeminal nerve, lens of the eye and in the pharyngeal arches. ALCAM has been analyzed via qRT-PCR and its transcription is downregulated just prior to the observed defasciculation phenotype. The pattern of expression of these genes combined with known expression of NT-3 receptors allows us to suggest a model whereby RA signaling regulates Sema4B, ALCAM and NT-3, which support the survival, guidance and fasciculation of the trigeminal nerve. This work has the potential to better understanding of the complex nature of cranial nervous system development.