Cell lines with the ability to differentiate into all types of somatic and germ cells represent a pluripotent developmental stage that transiently exists in vivo in the epiblast cells of the pre-implantation embryo. Given the lack of access to human neurons, together with the limited numbers and heterogeneity of neurons obtainable from rodent primary cultures, the directed differentiation of pluripotent cell lines into defined cells of the neural lineage has provided a novel versatile tool in neurobiology. Offering a potentially unlimited source of material, directed differentiation of pluripotent cell lines has been particularly well combined with high-throughput transcriptomic and epigenetic analyses. Here, we first overview the potential of different pluripotent lines to give rise to different types of neurons. Then, we discuss the emerging use of neuronal differentiation systems as a tool for unravelling mechanisms that regulate neuronal development and specification, modelling complex neurological diseases and understanding neuronal dysfunction. Beyond providing original insights in many aspects of neuronal biology, these tools have greatly facilitated the development of novel therapeutic interventions for neurological disorders.