BET domain epigenetic reader proteins play a role in controlling the expression of genes that regulate cellular differentiation and proliferation and phenotype maintenance (e.g., cMyc) and are frequently implicated in cancer; hence BET modulators are being explored as potential therapeutics in a number of tumour settings. In addition to regulating tumour cell viability, BET inhibitors are also associated with gastrointestinal toxicity, though the molecular and cellular mechanisms behind this toxicity have yet to be elucidated. We have found that BET inhibitors induce a dose-limiting duodenal villous atrophy in vivo, accompanied by inappetance and body weight loss. Ex-vivo cultures of intestinal organoids confirm that villous atrophy occurs with multiple chemical classes of BET domain inhibitors and that their toxicity is driven by their primary pharmacology. Intriguingly, transcriptomic analysis of the intestinal atrophy occurs in the absence of a proliferative block, and in the presence of cMyc, suggesting that the intestinal effects are not mediated by cMyc inhibition as may have been assumed. We find instead that BET domain inhibitors induce a rapid loss of fast cycling intestinal stem cells, suggesting that it is a loss of stem-cell self-renewal leading to crypt loss and dose-limiting duodenal toxicity. An inter-species comparison of the in vitro miniGut toxicity data indicated that human organoids are markedly less-sensitive to BET domain inhibitors mediated toxicity compared to other preclinical species (mouse, rat and dog).