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Background Environmental stresses harbored by the tumor microenvironment (TME) impose substantial barriers to cancer treatment. Many solid malignancies create a hyperosmotic TME due to increased interstitial fluid pressure from abnormal lymphatic drainage and excessive extracellular matrix deposition. Recent studies show that TME-induced activation of the tonicity-responsive transcription factor NFAT5 suppresses CD8+ T cell effector functions in vivo. Intriguingly, progenitor-like exhausted T cells (Tpex) harbored higher levels of NFAT5 than terminally exhausted T cells (Ttex), suggesting a potential link between osmotic stress and T cell differentiation, which is investigated in this study.Methods To evaluate the effect of osmotic stress on T cell differentiation, we screened a panel of osmolytes using an in vitro exhaustion assay. N-acetylneuraminic acid (Neu5Ac), the predominant sialic acid in human, stood out as a unique regulator enhancing T cell expansion while inhibiting differentiation. Neu5Ac-treated T cells were evaluated for their function and therapeutic efficacy in multiple adoptive transfer models, including chronic infection, syngeneic, and xenograft mouse tumors. Transcriptomics and metabolomics analyses were performed to elucidate the underlying mechanisms.Results Our osmolyte screening revealed that many solutes, ionic or non-ionic, inhibited T cell differentiation and proliferation when added at hyperosmotic concentrations. Uniquely, Neu5Ac simultaneously promoted T cell expansion while suppressing terminal differentiation, through facilitating the expansion of progenitor-like T cells and reprogramming T tex into less exhausted states. In adoptive transfer therapy models, Neu5Ac-pretreated T cells displayed superior in vivo proliferation, persistence, and tumor control. Mechanistically, activation of NFAT5, the only identified tonicity-responsive transcription factor, is required to suppress T cell terminal differentiation in vitro. Hyperosmolarity-activated NFAT5 led to the inhibition of AKT-mediated FOXO1 phosphorylation, thus upregulating the expression of T cell stemness- and memory-related genes controlled by FOXO1. Compared to other osmolytes, Neu5Ac also enhanced mitochondria fitness and promoted antioxidant production, resulting in reduction of cellular reactive oxygen species (ROS): while this effect played a lesser role in maintaining T cell stemness, it primarily enhanced T cell metabolic fitness and proliferation, distinguishing Neu5Ac as a unique osmolyte capable of decoupling T cell proliferation from differentiation.Conclusions Our study uncovered the fundamental phenomenon that hyperosmolarity promotes the maintenance of T cell stemness, providing new insights into how environmental stress contributes to regulation of T cell functions. Furthermore, Neu5Ac is identified as a unique osmolyte that uncouples T cell differentiation from proliferation, offering a viable strategy to generate stem-like T cells for enhancing adoptive cell therapies.Ethics Approval All mice experiments and procedures conducted were approved by The Institutitonal Animal Care and Use Committee (IACUC) at The Scripps Research Institute (TSRI), in accordance with NIH guidelines. De-identified human blood samples were purchased from Scripps Normal Blood Donor Service (NBDS) provided by the Scripps General Clinical Research Center (GCRC), with a lab Institutional Review Board (IRB) protocol approved by the Scripps Human Subjects Committee (HSC) for normal blood drawing. Human head and neck squamous cell carcinoma samples were de-identified and provided by Dr. J. Silvio Gutkind and Dr. Robert Saddawi-Konefka at UC San Diego Moores Cancer Center, La Jolla, through the College of American Pathologists (CAP)-accredited UC San Diego (UCSD) Biorepository and Tissue Technology Shared Resources (BTTSR). The collection and use of these samples were approved by IRB at UCSD and TSRI.