The guidance of axonal projections to ipsilateral and contralateral regions is essential for integration of bilateral sensory information and coordination of movement. In the development of olivocerebellar projections, newborn neurons of inferior olivary (IO) nuclei ventrally migrate from the hindbrain rhombic lip to the floor plate (FP). The cell bodies of IO neurons cannot cross the FP but their axons can, and thus IO neurons project their axons only to the contralateral cerebellar cortex. The molecular mechanisms determining the contralateral axonal projections of IO neurons, however, are obscure. The IO neurons and their axons express EphA4, whereas the FP expresses an EphA4 ligand, EphrinB3, from embryonic day 12.5. Therefore, we tested whether EphA4-deficient mice (EphA4-/-) would show impairment in the development of olivocerebellar projections. We found that, in EphA4-/- embryos, some of the IO neurons projected their axons to the ipsilateral cerebellar cortex because the cell bodies of the IO neurons abnormally crossed the FP. Furthermore, even in adults, EphA4-/- cerebella were bilaterally innervated by unilateral IO subnuclei. These observations indicate that EphA4 is involved in the contralateral axonal projections of IO neurons by preventing their cell bodies from crossing the midline FP. J. Comp. Neurol. 520:17021720, 2012. (C) 2011 Wiley Periodicals, Inc.