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Gut hormones secreted by enteroendocrine cells (EECs) coordinate the postprandial response to a meal by regulating digestion, absorption, circulating nutrient availability and satiety. EECs are specialised endocrine cells scattered throughout the gastrointestinal epithelium that release hormones in response to a wide range of luminal and systemic signals. This thesis focused on characterising the secretory mechanisms of three important gut hormones: glucagon-like peptide 1 (GLP-1, which has potent insulinotropic and anorexigenic effects), motilin (a key regulator of gut motility which is not expressed in rodent models) and serotonin (5-HT, a monoamine neurotransmitter with wide-ranging effects within and outside the GI tract). Several in vitro models have been developed to study the mechanisms of EEC regulation; however, at the outset of this project, it was not possible to identify human EECs without prior fixation and immunostaining which precluded functional studies. We therefore used CRISPR-Cas9-mediated knock-in to generate human intestinal organoids with fluorescently labelled EECs. Organoids are three dimensional self-renewing cultures grown from the adult stem cells of intestinal crypts which express mature epithelial cell types, including EECs. The properties of labelled human organoid EECs were investigated using a combination of fluorescence-activated cell sorting (FACS), bulk RNA sequencing, gut hormone secretion assays, single cell Ca²⁺ imaging, immunohistochemistry and peptidomics. GLU-Venus ileal organoids were first used to explore the mechanisms regulating GLP-1 release from human L-cells. We optimised protocols for the study and differentiation of EECs in human organoid culture, and used these to determine that human L-cells are highly phenotypically similar to their murine counterparts. GLP-1 secretion was stimulated by glucose, angiotensin II, arginine vasopressin and synthetic agonists of receptors for bile acids (GPBAR1), long chain fatty acids (FFA1) and monoacylglycerols (GPR119). Bulk RNA sequencing performed on human L-cells will guide future investigations. We next generated MLN-Venus and MLN-GCaMP7s duodenal organoids, and used these to carry out the first in-depth transcriptomic and functional characterisation of human motilin-expressing M-cells. Several receptors important for the postprandial and interdigestive regulation of motilin release were identified, including GPBAR1, FFA1 and GPR119. Acidification also stimulated acute M-cell activation, which was dependent on acid-sensing ion channels (ASICs), and ASIC-independent motilin secretion. To study 5-HT secretion from intestinal enterochromaffin cells (ECs), organoids with labelled tryptophan hydroxylase 1 expressing cells (TPH1-Venus) were established from human duodenum, ileum and rectum. Bulk RNA sequencing was performed on duodenal ECs, and considered in parallel with single cell RNA sequencing data of small intestinal EECs (isolated based on chromogranin A expression). Several highly enriched receptors were identified and initial Ca²⁺ imaging data suggested a possible stimulatory role for the short chain fatty acid acetate and the aromatic amino acid tryptophan in some duodenal ECs. Finally, we developed new methods and tools which are expected to prove useful for future studies of human EEC function. Organoids expressing an L-cell localised cyclic AMP sensor will enable intracellular responses to a wider range of stimuli to be monitored. We also established robust protocols to knockout genes of interest in human organoid cultures, which will allow for the detailed investigation of factors regulating EEC activation and differentiation. In summary, this thesis has demonstrated the utility of CRISPR-Cas9-modified intestinal organoid models for the study of stimulus-secretion coupling in human EECs, and opened several new avenues for further investigation. We hope that the insights obtained will improve our understanding of enteroendocrine physiology and the role of gut hormone therapies in the treatment of type 2 diabetes, obesity and motility disorders.