This article describes the derivation of midbrain dopamine (DA) neurons from human embryonic stem (hES) cells. The study outlines a method to direct hES cells into neurons of midbrain DA identity by using stromal feeder cells and sequential application of defined patterning molecules that mimic in vivo midbrain development. The process involves neural induction through stromal feeder cells, followed by regional specification using factors that direct midbrain development. The progression toward a midbrain DA neuron fate was monitored through the expression of key transcription factors, DA release measurements, tetrodotoxin-sensitive action potentials, and electron-microscopic visualization of tyrosine-hydroxylase-positive synaptic terminals. The high-yield derivation of DA neurons was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons represents a significant step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to study the molecular mechanisms controlling the development and function of human midbrain DA neurons. The study highlights the importance of stromal feeder cells in neural induction and the role of specific signaling molecules in directing the differentiation of hES cells into midbrain DA neurons. The findings demonstrate that pathways important for in vivo midbrain development can be systematically applied to direct hES cell differentiation into DA neurons in vitro. The study also shows that the differentiation of hES cells into midbrain DA neurons is highly efficient and reproducible across various hES and monkey ES cell lines. The results suggest that the derived DA neurons express the full complement of midbrain DA neuron markers and exhibit in vitro functionality, providing a basis for assessing the therapeutic potential of hES cells in preclinical models of Parkinson's disease. The study also demonstrates that the derived DA neurons can be used for mechanistic studies on human midbrain DA neuron development. The study concludes that the derivation of midbrain DA neurons from hES cells is a promising approach for regenerative medicine and provides a valuable tool for understanding the development and function of human midbrain DA neurons.This article describes the derivation of midbrain dopamine (DA) neurons from human embryonic stem (hES) cells. The study outlines a method to direct hES cells into neurons of midbrain DA identity by using stromal feeder cells and sequential application of defined patterning molecules that mimic in vivo midbrain development. The process involves neural induction through stromal feeder cells, followed by regional specification using factors that direct midbrain development. The progression toward a midbrain DA neuron fate was monitored through the expression of key transcription factors, DA release measurements, tetrodotoxin-sensitive action potentials, and electron-microscopic visualization of tyrosine-hydroxylase-positive synaptic terminals. The high-yield derivation of DA neurons was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons represents a significant step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to study the molecular mechanisms controlling the development and function of human midbrain DA neurons. The study highlights the importance of stromal feeder cells in neural induction and the role of specific signaling molecules in directing the differentiation of hES cells into midbrain DA neurons. The findings demonstrate that pathways important for in vivo midbrain development can be systematically applied to direct hES cell differentiation into DA neurons in vitro. The study also shows that the differentiation of hES cells into midbrain DA neurons is highly efficient and reproducible across various hES and monkey ES cell lines. The results suggest that the derived DA neurons express the full complement of midbrain DA neuron markers and exhibit in vitro functionality, providing a basis for assessing the therapeutic potential of hES cells in preclinical models of Parkinson's disease. The study also demonstrates that the derived DA neurons can be used for mechanistic studies on human midbrain DA neuron development. The study concludes that the derivation of midbrain DA neurons from hES cells is a promising approach for regenerative medicine and provides a valuable tool for understanding the development and function of human midbrain DA neurons.