The Midline Glia of Drosophila are required for nervous system morphogenesis and midline axon guidance during embryogenesis. In origin, gene expression and function, this lineage is analogous to the floorplate of the vertebrate neural tube. The expression or function of over 50 genes, summarised here, has been linked to the Midline Glia. Like the floorplate, the cells which generate the Midline Glia lineage, the mesectoderm, are determined by the interaction of ectoderm and mesoderm during gastrulation. Determination and differentiation of the Midline Glia involves the Drosophila EGF, Notch and segment polarity signaling pathways, as well as twelve identified transcription factors. The Midline Glia lineage has two phases of cell proliferation and of programmed cell death. During embryogenesis, the EGF receptor pathway signaling and Wrapper protein both function to suppress apoptosis only in those MG which are appropriately positioned to separate and ensheath midline axonal commissures. Apoptosis during metamorphosis is regulated by the insect steroid, Ecdysone. The Midline Glia participate in both the attraction of axonal growth cones towards the midline, as well as repulsion of growth cones from the midline. Midline axon guidance requires the Drosophila orthologs of vertebrate genes expressed in the floorplate, which perform the same function. Genetic and molecular evidence of the interaction of attractive (Netrin) and repellent (Slit) signaling is reviewed and summarised in a model. The Midline Glia participate also in the generation of extracellular matrix and in trophic interactions with axons. Genetic evidence for these functions is reviewed.