Embryonic stem cells (ESCs) are self‐renewing pluripotent cells derived from the inner cell mass (ICM) of blastocyst of the developing embryo. This chapter focuses on the use of (human) pluripotent stem cells (PSCs) and their progeny to assess (developmental) neurotoxicity. The neural tube is formed by neuroepithelial progenitor (NEP) cells. These primary neural stem cells are the origin of nearly all neurons and glial cells of the brain and spinal cord. Since the past few years, human stem cells (hSCs) are a promising source of human cells for mechanistically oriented developmental neurotoxicity /neurotoxicity (DNT/NT) safety assessment. Common methods to assess developmental neurotoxicity rely on high‐dose testing in experimental animals. To replace the traditional animal‐based tests, alternative in vitro test systems require a high predictivity of adverse neurotoxic effects in the (developing) brain. During neurodevelopment, the process of neurogenesis and gliogenesis occurs highly coordinated directly after neural tube closure.