ESCRT proteins were initially isolated in yeast as a single functional set of conserved components controlling endosomal cargo sorting and multivesicular body (MVB) biogenesis. Recent work has suggested that metazoan ESCRT proteins might have more functionally diverse roles, but the limited availability of ESCRT mutants in species other than yeast has hampered a thorough analysis. Here, we used a genetic screening strategy based on both cell-autonomous and non-autonomous growth-promotion phenotypes to isolate null mutations in nearly half of the ESCRT-encoding genes of Drosophila, including components of ESCRT-I, ESCRT-II and ESCRT-III complexes. All ESCRT components are required for trafficking of ubiquitylated proteins and are required to prevent excess Notch and EGFR signaling. However, cells lacking certain ESCRT-III components accumulate fewer ubiquitylated molecules in endosomes and display reduced degrees of cell proliferation compared with those lacking components of ESCRT-I and ESCRT-II. Moreover, although we find by ultrastructural analysis that MVB formation is impaired in ESCRT-I and ESCRT-II mutant cells, MVB biogenesis still occurs to some degree in ESCRT-III mutant cells. This work highlights the multiple cell biological and developmental roles of ESCRT proteins in Drosophila, suggests that the metazoan ESCRT-I, ESCRT-II and ESCRT-III complexes do not serve identical functions, and provides the basis for an extensive analysis of metazoan ESCRT function.