Two important features that characterize nervous system development are the generation of distinct neuronal subtypes and the establishment of precise connections between them. Previous studies in Drosophila and vertebrates have shown that the acquisition of specific neuronal identity requires concerted actions of multiple transcription factors during animal development. However, elucidating the molecular mechanisms that are governed by these transcription factors has been difficult, partly due to the lack of definitive molecular markers that allow unambiguous identification of neuronal fates. The olfactory system of Drosophila offers an excellent model to address this problem, as it is comprised of ~60 olfactory sensory neuron (OSN) subtypes; almost all of these are defined by the expression of a single, distinct ligand-specific olfactory receptor gene. These diverse OSNs arise from a seemingly homogenous population of progenitor cells located at distinct regions in the antennal imaginal disc. These properties allow us to assay for the spatial and temporal contributions of the developmental determinants that are involved in specifying and diversifying OSN fate. Using a transgenic RNAi approach, we have tested the role of 808 genes in antennal OSN specification, encoding putative transcription factors, chromatin regulators and embryonic patterning determinants. To circumvent the detrimental effects of ubiquitous gene knock-down, we induced RNAi specifically in the eye-antennal discs from the second instar larval stage onwards using a combination of an actin-flip out-GAL4 driver and eyeless-Flippase. Correct OSN specification entails both the expression of a particular olfactory receptor gene and targeting of its axon to a specific glomerulus in the antennal lobe. We therefore screened for phenotypic defects upon gene knock-down by examining the expression of olfactory receptor reporters and axon projections in two different populations of OSNs, as well as the global structural integrity of antennal lobe glomeruli. Our primary screen has revealed that ~15% of the tested genes might play a role in OSN specification and/or development. In an attempt to elucidate the processes that are regulated by these genes during olfactory system development, these genes were further classified into different categories based on their RNAi phenotype (eye-antennal defects, antennal lobe malformation, ectopic structures, reduced axon density, axonal mistargeting and other defects). The majority of genes (>82%) exhibit multiple phenotypic defects upon RNAi, suggesting that these factors could regulate different functions, depending on their spatial and temporal contexts. We will present the results of this screen and our follow-up work on individual determinants of OSN identity and wiring.