To fully realize the potential of human pluripotent stem cells (hPSCs) for both therapeutic and research purposes, it is critical to follow an efficient and reliable in vitro differentiation method that is based on optimal physical, chemical and developmental cues. This highly reproducible protocol describes how to grow hPSCs such as human induced pluripotent and embryonic stem cells in a physically confined area (‘spot’) and efficiently differentiate them into a highly enriched population of healthy and functional midbrain dopamine progenitors (mDAPs) and midbrain dopamine neurons (mDANs). The protocol takes 28 d, during which cells first grow and differentiate in spots for 14 d and then are replated and further differentiated for a further 14 d as a monolayer culture. We describe how to produce mDAPs, control the quality of cells and cryopreserve mDAPs without loss of viability. Previously we showed that mDANs generated by this ‘spotting’-based method exhibit gene expression and (electro)physiological properties typical of A9 mDANs lost in Parkinson’s disease brains and can rescue motor defects when transplanted into the striatum of 6-hydroxydopamine-lesioned rats. This protocol is scalable for production of mDAPs under good manufacturing practice conditions and was also previously successfully used to generate cells for the first autologous cell replacement therapy of a patient with Parkinson’s disease without the need for immune suppression. We anticipate this protocol could also be readily adapted to use spotting-based culture to further optimize the differentiation of hPSC to alternative differentiated cell types.