Notch signaling is involved in numerous cellular processes during development and throughout adult life. Although ligands and receptors are largely expressed in the whole organism, activation of Notch receptors only takes place in a subset of cells and/or tissues and is accurately regulated in time and space. Previous studies have demonstrated that endocytosis and recycling of both ligands and/or receptors are essential for this regulation. However, the precise endocytic routes, compartments and regulators involved in the spatio temporal regulation are largely unknown.In order to identify Notch signaling intracellular trafficking regulators, we have undertaken a tissue-specific dsRNA genetic screen against candidates potentially involved in endocytosis and recycling within the endolysosomal pathway. dsRNA against 418 genes was induced in Drosophila melanogaster sensory organ lineage in which Notch signaling regulates binary cell fate acquisition. Gain- or loss-of Notch signaling phenotypes were observed in adult sensory organs for 113 of them. Furthermore, 26 genes presented a change in the steady state localization of Notch, Sanpodo, a Notch co-factor, and/or Delta in the pupal lineage. In particular, we identified 20 genes with previously unknown function in Drosophila melanogaster intracellular trafficking. Among them, we identified CG2747 and show that it regulates the localization of clathrin adaptor AP-1 complex, a negative regulator of Notch signaling. All together, our results further demonstrate the essential function of intracellular trafficking in regulating Notch signaling-dependent binary cell fate acquisition and constitute an additional step toward the elucidation of the routes followed by Notch receptor and ligands to signal.