For cell replacement therapy of neurodegenerative diseases such as Parkinson's disease (PD), methods for efficiently generating midbrain dopaminergic (DA) neurons from embryonic stem (ES) cells have been investigated. Two aspects of DA neuron generation are considered: genetic modification and manipulation of culture conditions. A transcription factor known as critical for development of DA neurons, Nurr1, was introduced into ES cells to see how they facilitate the generation of DA neurons from ES cells. Also, two culture procedures, the 5-stage method and stromal cell-derived inducing activity (SDIA) method, were used for ES cell differentiation. Using the 5-stage method, we and others previously demonstrated that Nurr1-overexpressing ES cells, under treatment of signaling molecules such as SHH and FGF8 followed by treatment of ascorbic acid, can differentiate into DA neurons with a high efficiency (> 60% of TH⁺/Tuj1⁺ neurons). Furthermore, using the SDIA method with treatment of signaling molecules, we found that Nurr1-overexpressing ES cells can differentiate to DA neurons with the highest efficiency ever reported (~90% of TH⁺/Tuj1⁺ neurons). Importantly, our semi-quantitative and real-time PCR analyses demonstrate that all known DA marker genes (e.g., TH, AADC and DAT) were up-regulated in Nurr1-overexpressing ES cells when compared to the naïve ES cells. These cells produced increased dopamine compared to naïve D3 cells after differentiation. In the in vivo context after transplantation, the genetically modified ES cells also showed the highly increased dopaminergic neuronal phenotypes. Thus, the combination of genetic engineering and appropriate culture conditions provides a useful tool to generate a good cell source from ES cells for cell replacement therapy of degenerative diseases such as PD.