Here, we present a protocol for differentiating human embryonic stem cells into adrenal chromaffin-like cells that express catecholaminergic markers (TH, DβH, PNMT, and CgA) and exhibit excitability and Ca2+ signaling. We describe the key steps for forming embryoid bodies (EBs) and subsequently directing them toward caudal neural commitment via Wnt/TGF-β regulation, followed by neural crest cell specification through BMP2 and FGF2 exposure, differentiation into chromaffin-like cells, and finally extended culture with BMP4 and glucocorticoids for physiological maturation.