Embryonic stem cells (ESCs) possess remarkable potential for regenerative medicine due to their unique ability to differentiate into various cell types of the body. Understanding the molecular signaling pathways and epigenetic regulation governing ESC fate determination is essential for harnessing their therapeutic potential. This review synthesizes current knowledge on the intricate interplay between molecular signaling pathways and epigenetic modifications in maintaining pluripotency and directing differentiation in ESCs. Pluripotency maintenance in ESCs is orchestrated by a network of signaling pathways, including the canonical Wnt/β-catenin, Sonic Hedgehog (SHH), and TGF-β/Activin/Nodal pathways, which converge on key transcription factors such as Oct4, Sox2, and Nanog. These factors form a core regulatory circuitry essential for sustaining ESC identity. Additionally, the balance between self-renewal and differentiation is delicately regulated by the interplay of various signaling cascades, ensuring the dynamic equilibrium of pluripotency. Upon induction of differentiation, ESCs undergo lineage-specific fate determination orchestrated by precise activation and repression of gene expression programs. Signaling pathways guide ESCs towards different germ layers, including endoderm, mesoderm, and ectoderm, through sequential activation of lineage-specific transcription factors. Moreover, epigenetic modifications, such as chromatin remodeling and histone modifications, play pivotal roles in modulating gene expression patterns during differentiation.