Receptor Tyrosine Kinase (RTK) signaling pathways control key cellular processes such as proliferation, migration and cell fate specification, in normal development and in disease. RTK pathways signal through an intracellular cascade of kinases, culminating in the phosphorylation and activation of the downstream effector kinase, MAPK/Erk. Once active, MAPK/Erk enters the nucleus where it phosphorylates transcriptional regulators, thereby modifying their function. This brings about coordinated changes in gene expression profiles that are imperative for subsequent cellular decisions. Despite the critical roles played by RTK pathways in various developmental processes, to date only a few confirmed transcription factors, which are directly targeted by Drosophila MAPK/Erk, are known. We have, therefore, established a genome-wide proteomics screen in order to uncover new direct nuclear MAPK/Erk substrates. So far, our assay has identified 35 putative targets for MAPK/Erk, some of which have been previously reported (e.g., Bicoid). For several newly selected MAPK/Erk targets, we have generated transgenic flies expressing unphosphorylatable as well as phosphomimetic derivatives. This approach will help validate bona fide MAPK/Erk-regulated targets, since expression of these variants is expected to exert differential outcomes in vivo. Recognizing novel MAPK/Erk substrates should ultimately enhance our understanding of how RTK signaling pathways induce specific cellular responses during development.