CO(2) sensation represents an interesting example of nervous system and behavioral evolutionary divergence. The underlying molecular mechanisms, however, are not understood. Loss of microRNA-279 in Drosophila melanogaster leads to the formation of a CO(2) sensory system partly similar to the one of mosquitoes. Here, we show that a novel allele of the pleiotropic transcription factor Prospero resembles the miR-279 phenotype. We use a combination of genetics and in vitro and in vivo analysis to demonstrate that Pros participates in the regulation of miR-279 expression, and that reexpression of miR-279 rescues the pros CO(2) neuron phenotype. We identify common target molecules of miR-279 and Pros in bioinformatics analysis, and show that overexpression of the transcription factors Nerfin-1 and Escargot (Esg) is sufficient to induce formation of CO(2) neurons on maxillary palps. Our results suggest that Prospero restricts CO(2) neuron formation indirectly via miR-279 and directly by repressing the shared target molecules, Nerfin-1 and Esg, during olfactory system development. Given the important role of Pros in differentiation of the nervous system, we anticipate that miR-mediated signal tuning represents a powerful method for olfactory sensory system diversification during evolution.