T lymphocytes, a key arm of adaptive immunity, are known to dynamically regulate O-glycosylation during T cell maturation and when responding to stimuli; however, the direct role of O-glycans in T cell maturation remains largely unknown. Using a conditional knockout of the gene (C1GalT1C1 or Cosmc) encoding the specific chaperone Cosmc, we generated mice whose T cells lack extended O-glycans (T cell conditional Cosmc knock out or TCKO mice) and homogeneously express the truncated Tn antigen. Loss of Cosmc is highly deleterious to T cell persistence, with near-complete elimination of Cosmc-null T cells from spleen and lymph nodes. Total T cell counts are 20% of wild type (WT), among which only 5% express the truncated glycans, with the remaining 95% consisting of escapers from Cre-mediated recombination. TCKO thymocytes were able to complete thymic maturation but failed to populate the secondary lymphoid organs both natively and upon adoptive transfer to WT recipients. Our results demonstrate that extended O-glycosylation is required for the establishment and maintenance of the peripheral T cell population.