Enteroendocrine cells (EECs) are a hormone-/neurotransmitter-producing population with well-defined physiological roles. Knowledge regarding their differentiation program in the human gut, however, is scarce. Deciphering endocrine specification could identify targets which allow the manipulation of specific EEC populations and form the basis for new treatments for metabolic, inflammatory and cognitive disorders. Isoxazole-9 (ISX-9) is a small molecule, previously used in protocols for neuronal and beta-cell differentiation. These cell types are developmentally similar to EECs and share lineage specification traits. In neuronal progenitors ISX-9 activates NeuroD1, a transcription factor that directs secretory differentiation in the gut. We explored the effect of ISX-9 on EEC identity in organoids derived from human tissue resident stem cells. ISX-9 promoted upregulation of NeuroD1, as expected, in addition to EEC progenitor marker Neurogenin 3 (Ngn3). Furthermore, an increase in the expression of Pax4, a known regulator of EEC differentiation in mouse, was also observed. Enterochromafin (chromogranin A (ChgA), TAC1, TPH1) and I-cell (cholecystokinin (CCK)) lineage markers were preferentially upregulated, whilst markers of other EEC lineages were either unaffected or down regulated. Immunostaining for ChgA and 5-HT and secretion assays confirmed functional enterochromaffin cell enrichment. An inducible Pax4-overexpressing human organoid model was generated to determine if this was sufficient to recreate the ISX-9 induced phenotype. Physiological levels of Pax4 expression recapitulated the phenotype exhibited by ISX-9. Interestingly high levels of expression inhibited all EEC differentiation and trapped cells in an early progenitor like state. These studies highlight the similarities between homeostatic mouse and human EEC specification and provides proof-of-concept that manipulating specific EEC cell populations is possible with small molecules. The potential to treat serotonin deficient diseases, eg depression by manipulating EEC cell fate is an attractive and novel supposition. Whether this can impact physiology and is translatable to the clinic remains to be determined.