Derivation of midbrain dopaminergic (DA) neurons from human embryonic stem (hES) cells has been of particular interest because of the clinical potential for DA neuron transplantation in patients with Parkinson's disease (PD). Several protocols for DA neuron differentiation from mouse embryonic stem cells and hES cells have been reported: however, protocols involving hES cells have yet to be improved. Here, we used a slightly modified stromal cell-derived inducing activity method, consisting four different culture stages, to show that KhES-1 cells differentiate into tyrosine hydroxylase (TH)-positive DA neurons. Quantitative real-time PCR analysis showed a marked induction of the DA neuron marker genes NURR1, paired-like homeodomain transcription factor 3 (PITX3), LIM homeobox transcription-factor 1, beta (LMX1B), engrailed-1 (EN1), dopamine transporter (DAT, and aromatic amino acid decarboxylase (AADC) during differentiation. Treatment with fibroblast growth factor (FGF)-20 and FGF-2 at the final differentiation stage induced the increase of DA neuron development-related transcription factors such as NURR1, PITX3, LMX1B, and EN1. FGF-20 and FGF-2 enhanced DA neuron differentiation from hES cell-derived neural progenitor cells directly without any soluble factors from PA6 cells. These results provide valuable information that will assist in efficient DA neuron differentiation from hES cells and for future transplant application. (c) 2009, The Society for Biotechnology, Japan. All rights reserved.