We have used enhancer traps and antibodies as markers of cell identity to assess the relative contribution of individual mesectodermal cell (MEC) lineages to CNS midline morphogenesis in four mutations that disrupt commissure formation in Drosophila. The absence of commissures, leading to longitudinal tract collapse, was seen in embryos mutant for the genes single-minded and slit. MEC lineages did not survive in single-minded mutant embryos, in contrast to the survival of all MEC lineages in slit mutant embryos. The midline glial cells were displaced and appeared ultrastructurally normal in slit mutant embryos, yet the presence of the MG was not sufficient to generate commissures. Commissure formation requires correct MEC cytoarchitecture, dependent upon slit activity. In fused commissure mutants (rhomboid and Star) neuron number was reduced in the ventral unpaired median neuron (VUM) lineage and the median neuroblast lineage before commissure formation (stage 12). Subsequent to these neuronal defects, the midline glia died by apoptosis (stage 13). Commissure fusion and glial apoptosis may be triggered by the earlier perturbations in MEC neuronal lineages. These studies establish when the respective activities of each gene are required for the development of each MEC lineage.