The Drosophila CNS midline cells develop from a homogenous mesectoderm into a variety of neurons and glia. These cells play essential roles in establishing the Drosophila CNS. Though relatively few in number, Drosophila CNS midline cells express a broad array of conserved neurotransmitters, including dopamine, glutamate, octopamine, and serotonin. Previous work on this system focused on generating a compendium of gene expression data that allowed for the unambiguous identification of each cell in the midline. My long-term goal is to use expression maps created from this analysis to identify mechanisms that generate diversity in the Drosophila midline. Several transcriptional pathways have already been shown to play a role in specifying midline cell fate. For instance, Notch signaling in the midline is both necessary and sufficient to generate glia. However, the pathways that generate glial diversity were only recently discovered. My work has shown that two transcription factors, runt and en, specify midline glial fate, and that hh, secreted from the epidermis, plays an active role in this process by acting upstream of en. These data highlight the importance of segmentation genes in specifying midline glia. In neurons, three segmentation genes are expressed in patterns that are similar to the known midline precursor (MP) domains: runt in the anterior, slp medially, and en in the posterior. I propose to determine the role of slp and en in specifying medial and posterior MP fate. To accomplish this goal, I will use the Gal4 system to ectopically express various negative regulators of slp and en in order to gauge the necessity of each transcription factor in MP specification. I will compare these results to mutant phenotypes for both transcription factors. In addition to these experiments, I will ectopically express slp and en throughout the midline in order to determine sufficiency. This work will establish the role of these transcription factors in MP and ultimately neuron fate. Further, I will identify the transcription factors that act in concert with a second wave of Notch signaling to specify neuron cell fate. This work will focus on the role en plays in establishing GABAergic iVUMs and inhibiting the formation of two glutamatergic cell types, H-cell sib and mVUMs. Additional work will focus on a number of transcription factors shared between H-cell sib and iVUMs, and their role in specifying aspects of these Notch-dependent cell types. Finally, I will examine the VGlut locus and identify regulatory sites that differentiate VGlut expression in iVUMs, H-cell sib, and mVUMs.