Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8 + surface proteome (surfaceome). Surprisingly, coculturing primary CD8 + T cells with Tregs only modestly affected the CD8 + surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8 + surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4 + T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function., Competing Interests: Conflict of interest A. A. is a cofounder of Tango Therapeutics, Azkarra Therapeutics, Ovibio Corporation; a consultant for SPARC, Bluestar, ProLynx, Earli, Cura, GenVivo and GSK; a member of the Scientific Advisory Board of Genentech, GLAdiator, Circle and Cambridge Science Corporation; receives grant/research support from SPARC and AstraZeneca; holds patents on the use of PARP inhibitors held jointly with AstraZeneca, which he has benefitted financially (and may do so in the future). A. M. is cofounder, member of the Boards of Directors and member of Scientific Advisory Boards of Spotlight Therapeutics and Arsenal Biosciences. A. M. has served as an advisor to Juno Therapeutics, was a member of the Scientific Advisory Board at PACT Pharma and was an advisor to Trizell. A. M. has received honoraria from Merck and Vertex, a consulting fee from AlphaSights, and is an investor in and informal advisor to Offline Ventures. A. M. owns stock in Arsenal Biosciences, Spotlight Therapeutics and PACT Pharma. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem. J. A. W. is cofounder of Soteria Therapeutics, is on the Scientific Advisory Board of Jnana Therapeutics, Inception Therapeutics, IgGenix Inc, Red Tree Capital, Spotlight Therapeutics, Inzen Therapeutics, and receives research support from Bristol-Myers-Squibb, TRex Bio and Merck, Inc., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)