Na_v1.6 is the main sodium channel isoform at adult nodes of Ranvier. Here, we show that Na_v1.2 and its β2 subunit, but not Na_v1.6 or β1, are clustered in developing central nervous system nodes and that clustering of Na_v1.2 and Na_v1.6 is differentially controlled. Oligodendrocyte-conditioned medium is sufficient to induce clustering of Na_v1.2 α and β2 subunits along central nervous system axons in vitro. This clustering is regulated by electrical activity and requires an intact actin cytoskeleton and synthesis of a non-sodium channel protein. Neither soluble- or contact-mediated glial signals induce clustering of Na_v1.6 or β1 in a nonmyelinating culture system. These data reveal that the sequential clustering of Na_v1.2 and Na_v1.6 channels is differentially controlled and suggest that myelination induces Na_v1.6 clustering.