The two serotonergic HSN motor neurons of the nematode Caenorhabditis elegans innervate the vulval muscles and stimulate egg laying by hermaphrodites. By analyzing mutant and laser-operated animals, we find that both epithelial cells of the developing vulva and axons of the ventral nerve cord are required for HSN axonal guidance. Vulval precursor cells help guide the growth cone of the emerging HSN axon to the ventral nerve cord. Vulval cells also cause the two HSN axons to join the ventral nerve cord in two separate fascicles and to defasciculate from the ventral nerve cord and branch at the vulva. The axons of either the PVP or PVQ neurons are also necessary for the HSN axons to run in two separate fascicles within the ventral nerve cord. Our observations indicate that the outgrowth of the HSN axon is controlled in multiple ways by both neuronal and nonneuronal cells.