Nerve roots have specialized transition zones that permit axon extension but limit cell movement between the CNS and PNS. Boundary cap cells prevent motor neuron soma from following their axons into the periphery, thereby contributing to a selective barrier. Transition zones also restrict movement of glial cells. Consequently, axons that cross the CNS–PNS interface are insulated by central and peripheral myelin. The mechanisms that prevent the migratory progenitors of oligodendrocytes and Schwann cells, the myelinating cells of the CNS and PNS, respectively, from crossing transition zones are not known. Here, we show that interactions between myelinating glial cells prevent their movements across the interface. Usingin vivotime-lapse imaging in zebrafish we found that, in the absence of Schwann cells, oligodendrocyte progenitors cross ventral root transition zones and myelinate motor axons. These studies reveal that distinct mechanisms regulate the movement of axons, neurons, and glial cells across the CNS–PNS interface.