This study describes the derivation of midbrain dopamine (DA) neurons from human embryonic stem (hES) cells using a combination of stromal feeder cells and defined patterning molecules. The process involves neural induction through stromal feeder cells, followed by regional specification using factors that mimic in vivo midbrain development. Key transcription factors, such as Pax2, Pax5, and En1, as well as functional markers like DA release, tetrodotoxin-sensitive action potentials, and tyrosine-hydroxylase (TH)-positive synaptic terminals, were used to monitor the differentiation process. The derived DA neurons expressed a full complement of midbrain DA neuron markers and exhibited in vitro functionality, providing a valuable resource for preclinical models of Parkinson's disease. The study demonstrates that hES cells can be efficiently differentiated into midbrain DA neurons under specific conditions. The differentiation protocol involves the use of stromal feeder cells, such as MS5, which promote neural differentiation in hES cells. The process includes the formation of neuroepithelial structures called neural rosettes, which are then replated and exposed to factors like FGF8 and SHH to induce midbrain DA neuron fate. The derived neurons were highly functional, with a high percentage expressing TH, the rate-limiting enzyme in DA synthesis. The study also highlights the potential of this system for mechanistic studies on human midbrain DA neuron development. The availability of a large number of functional midbrain DA neurons opens new avenues for research and therapeutic applications, particularly in the context of Parkinson's disease. The protocol is highly reproducible across multiple hES and monkey ES cell lines, suggesting its broad applicability. However, further studies are needed to evaluate the long-term survival and maintenance of the derived neurons in vivo. The use of human feeder cells or feeder-free systems may be necessary for clinical applications to ensure safety and avoid potential immune rejection. The findings underscore the importance of understanding the molecular mechanisms underlying midbrain DA neuron development and function, which could lead to the development of novel therapies for neurodegenerative diseases.This study describes the derivation of midbrain dopamine (DA) neurons from human embryonic stem (hES) cells using a combination of stromal feeder cells and defined patterning molecules. The process involves neural induction through stromal feeder cells, followed by regional specification using factors that mimic in vivo midbrain development. Key transcription factors, such as Pax2, Pax5, and En1, as well as functional markers like DA release, tetrodotoxin-sensitive action potentials, and tyrosine-hydroxylase (TH)-positive synaptic terminals, were used to monitor the differentiation process. The derived DA neurons expressed a full complement of midbrain DA neuron markers and exhibited in vitro functionality, providing a valuable resource for preclinical models of Parkinson's disease. The study demonstrates that hES cells can be efficiently differentiated into midbrain DA neurons under specific conditions. The differentiation protocol involves the use of stromal feeder cells, such as MS5, which promote neural differentiation in hES cells. The process includes the formation of neuroepithelial structures called neural rosettes, which are then replated and exposed to factors like FGF8 and SHH to induce midbrain DA neuron fate. The derived neurons were highly functional, with a high percentage expressing TH, the rate-limiting enzyme in DA synthesis. The study also highlights the potential of this system for mechanistic studies on human midbrain DA neuron development. The availability of a large number of functional midbrain DA neurons opens new avenues for research and therapeutic applications, particularly in the context of Parkinson's disease. The protocol is highly reproducible across multiple hES and monkey ES cell lines, suggesting its broad applicability. However, further studies are needed to evaluate the long-term survival and maintenance of the derived neurons in vivo. The use of human feeder cells or feeder-free systems may be necessary for clinical applications to ensure safety and avoid potential immune rejection. The findings underscore the importance of understanding the molecular mechanisms underlying midbrain DA neuron development and function, which could lead to the development of novel therapies for neurodegenerative diseases.