Neuronal differentiation is a highly regulated process in which morphogens establish cellular identities through coordinated transcriptional programs. However, the contribution of physiologically derived metabolites to human neural development remains poorly understood. β-hydroxybutyrate (bhb) has been recognized as an inflammatory, epigenetic, energetic, and neuroprotective regulator. We differentiated midbrain floor plate neural precursor cells (mfNPC) from human embryonic stem cells to generate midbrain organoids (MBOs), which were exposed to bhb. Although dopaminergic differentiation occurred in control MBOs, treatment with 1 mM bhb for 16 days significantly increased both the number of Tyrosine hydroxylase-positive (TH⁺) neurons, and the corresponding transcripts assessed by bulk RNA sequencing. Such transcriptomic profiling revealed modest but consistent bhb-associated changes in trophic, neuronal and dopaminergic transcripts. To further characterize these changes, we performed gene set enrichment analysis using curated cell phenotypes-associated transcriptional programs. These analyses indicated that bhb treatment is associated with shifts in the enrichment scores of dopaminergic programs in day 16 MBOs. At the epigenomic level, we used CUT&RUN assays to map H3K27 acetylation, and the selected candidates H3K27ac regions after bhb treatment were linked to their closest neighboring genes, suggesting positive enrichment of neural and dopaminergic gene ontology categories at both time points. Unsupervised analysis of H3K27ac-enriched regions revealed three major clusters detected across differentiation, although present in both control and bhb-treated MBOs. Motif enrichment analysis of clustering regions identified distinct predicted transcription factor binding motifs in mfNPC and MBOs. Finally, we integrated this motif analysis, enhancer annotation, and our transcriptomic data on a web platform, Enhancer Network Explorer, to explore relationships between H3K27ac profiles, transcriptional changes and bhb exposure during human MBOs differentiation. Together, our results indicate that bhb treatment is associated with increased TH-positive cell abundance and with population-level changes related to neurodevelopmental transcriptional programs. These findings suggest that bhb might support early human midbrain differentiation and provide a starting point for future studies addressing the mechanisms linking metabolic cues, chromatin-associated regulation and dopaminergic differentiation.