Purpose: Proper control of eye movements is critical to vision, but relatively little is known about the molecular mechanisms that regulate development and axon guidance in the ocular motor system or cause the abnormal innervation patterns (oculomotor synkinesis) seen in developmental disorders and after oculomotor nerve palsy. We developed an ex vivo slice assay that allows for live imaging and molecular manipulation of the growing oculomotor nerve, which we used to identify axon guidance cues that affect the oculomotor nerve. Methods: Ex vivo slices were generated from E10.5 IslMN-GFP embryos and grown for 24 to 72 hours. To assess for CXCR4 function, the specific inhibitor AMD3100 was added to the culture media. Cxcr4cko/cko:Isl-Cre:ISLMN-GFP and Cxcl12KO/KO:ISLMN-GFP embryos were cleared and imaged on a confocal microscope. Results: When AMD3100 was added to the slice cultures, oculomotor axons grew dorsally (away from the eye) rather than ventrally (toward the eye). Axons that had already exited the midbrain continued toward the eye. Loss of Cxcr4 or Cxcl12 in vivo caused misrouting of the oculomotor nerve dorsally and motor axons from the trigeminal motor nerve, which normally innervate the muscles of mastication, aberrantly innervated extraocular muscles in the orbit. This represents the first mouse model of trigeminal-oculomotor synkinesis. Conclusions: CXCR4/CXCL12 signaling is critical for the initial pathfinding decisions of oculomotor axons and their proper exit from the midbrain. Failure of the oculomotor nerve to innervate its extraocular muscle targets leads to aberrant innervation by other motor neurons, indicating that muscles lacking innervation may secrete cues that attract motor axons.