Serotonin (5-HT) is a critical neurotransmitter involved in regulating mood, stress, and behavior. Synthesized by serotonergic neurons in the brainstem’s raphe nuclei, 5-HT maintains neural balance through interactions with various receptor subtypes. Dysfunctions in 5-HT pathways are linked to neuropsychiatric disorders like anxiety and depression. Recent discoveries have shown that 5-HT plays a central role in shaping the brain’s epigenetic landscape by covalently binding to histone tails, thereby modifying chromatin structure and influencing gene expression. This novel pathway differs from serotonin’s synaptic role and may affect long-term cellular functions and behavior. Notably, preliminary findings suggest serotonylation plays a key role in stress-mediated transcriptional response, with disruptions rescuing stress-induced gene expression and behavior in mice. Despite insights from animal models, the relevance of serotonylation to the human brain remains unexplored. This study aims to investigate the interaction between 5-HT, epigenetic mechanisms, and gene regulation in pluripotent stem cell-derived human raphe nucleus organoids (hRNO). We will determine baseline serotonylation levels, test how increased and reduced 5-HT levels affect chromatin and transcriptional dynamics and determine how these changes control their electrophysiological activity. The results of this study could pave the way for the development of new therapeutic strategies for treating stress-related disorders.