Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. ESCs have two distinctive properties: ability to proliferate indefinitely, a feature referred as “self-renewal”, and to differentiate into different cell types, a peculiar characteristic known as “pluripotency”. Self-renewal and pluripotency of ESCs are finely orchestrated by precise external and internal networks including epigenetic modifications, transcription factors, signaling pathways, and histone modifications. In this systematic review, we examine the main molecular mechanisms that sustain self-renewal and pluripotency in both murine and human ESCs. Moreover, we discuss the latest literature on human naïve pluripotency.