Aberrant expression of truncated mucin type O-glycans is a known hallmark of epithelial cancers and represents an attractive drug target due to the relative low expression of these glycoforms in normal human tissue. However, the underlying mechanisms resulting in truncation of O-glycans as well as their potential roles in cancer development has yet to be fully understood. To investigate O-glycan influence on tissue formation and cancer, we created a library of human, primary keratinocytes with genetically engineered deficiencies in O-glycosylation capacity. Using an organotypic 3D model of human skin, we show that induction of truncated O-glycans instigates delayed differentiation and change in tissue architecture in the basal layers. Furthermore, our genetically engineered cell-lines provide evidence that truncated O-glycans result in increased proliferation. To map the O-glycan landscape of human cancers in vivo, we conducted a systematic analysis of T-, Tn- and STn-antigen expression in several tissue samples from a range of epithelial cancers using immunohistochemistry. In order to probe the mechanisms underlying O-glycan truncation, we employed a strategy combining whole-genome, CRISPR-Cas mediated loss-of-function screening and flow cytometry sorting based on glycan-specific antibodies. This strategy yields a deeper understanding of the mutagenic landscape potentially resulting in O-glycan truncation in human cells (and potentially cancers), extending beyond perturbations of genes already known to results in O-glycan truncation, i.e. COSMC and C1GALT1. In conclusion, our work indicates potential roles for truncated O-glycans in tissue homeostasis, provides a mapping of O-glycoforms in human cancers and yields new insights into mechanisms underlying O-glycan truncation.