The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and wellstudied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventynine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system. KEYWORDS Drosophila; CNS development; neural cell fate specification; forward genetic screening; FMRFamide D URING nervous system development, a restricted number of progenitors generate the vast number of neurons and glia that build the mature central nervous system (CNS). The final identity of a specific neuron is dependent upon a complex series of regulatory steps, including spatial and temporal cues, asymmetric cell division, and terminal cell fate determinants (Allan and Thor 2015). In addition to the generation of a myriad of unique cell fates, each neural subtype furthermore is generated in precise numbers, and thus both proliferation and apoptosis are tightly regulated during development. In spite of tremendous progress during the last decades in deciphering these regulatory events, our understanding of how they integrate within the context of any specific neuronal lineage to ensure final cell specification and cell number is still fragmentary.The Drosophila melanogaster CNS can be subdivided into the brain and the ventral nerve cord (VNC). The VNC is formed from the ventral part of the neuroectoderm by highly conserved anterior-posterior and dorsal-ventral patterning of the embryo (Skeath 1999;Skeath and Thor 2003). The VNC can be subdivided into three thoracic and 10 abdominal segments (Birkholz et al. 2013). During early embryogenesis 30 progenitors, denoted neuroblasts (NBs), a...