The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Our recent work revealed that its lysosomal degradation relies upon the activity of the E1 ubiquitin ligase UBA1 and is dependent on ubiquitylation at lysine residues 203 and 213. The endosomal deubiquitylase, USP8, interacts with CTLA4, and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells, and cancer cell–derived exosomes. Re-expression of wild-type USP8, but neither a catalytically inactive nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4 or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover. To identify the specific ubiquitin E3 ligase(s) responsible for targeting CTLA4 degradation, we have engineered a stable cell line expressing SNAP-tagged CTLA4 that faithfully recapitulates the short-lived property of endogenous CTLA4. Using this system, we conducted a FACS-based pooled CRISPR/Cas9 screen of human E3 ubiquitin ligases, assessing surface CTLA4 levels using a fluorescent membrane-impermeable SNAP ligand, under both steady-state and post-endocytic conditions.