Several methods for producing dopaminergic (DA) neurons from human embryonic stem cells (ESC) are known; however, these methods are generally either complex, involve multiple steps and manual selection of cells, or require the use of animal cells or products. Stromal-derived inducing activity (SDIA) refers to the property of mouse stromal cells, especially the PA6 cell line, of inducing ESC to differentiate into DA neurons. Chemical duplication of SDIA could provide an alternate, and potentially simpler, method for inducing ESC to undergo DA differentiation. To identify the biochemical nature of SDIA, we carried out gene expression profiling of PA6 cells, comparing PA6 cells to cell lines which lack SDIA. Factors highly expressed by PA6 cells included stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). The combination of these factors was termed SPIE. In vitro functional analysis of candidate molecules on DA differentiation of hESC was carried out following a brief (2-4 day) embryoid body formation phase. After 10 days, the majority of colonies differentiated from the BG01V2 and BG03 hESC lines by exposure to SPIE contained numerous cells expressing the midbrain neural progenitor cell marker Msx1 and tyrosine hydroxylase (TH). To further characterize the role of each candidate molecule in TH+ cell induction, we individually excluded each factor from the culture medium. The absence of any one factor resulted in a substantial decrease in DA differentiation as compared to complete SPIE. Omission of SDF-1 or EFNB1 decreased the number of TH+ colonies by more than 50% in comparison to cultures exposed to SPIE. Cultures lacking PTN or IGF2 contained 33% and 41% fewer TH-expressing colonies respectively. In addition, numbers of TH+ cells within colonies were greatly decreased in conditions lacking any one of the factors. RT-PCR and western blot analysis confirmed the expression of midbrain specific markers, including engrailed 1, Nurr1, Pitx3, and dopamine transporter in cells exposed to SPIE. SPIE-induced DA neurons generated action potentials and formed functional synaptic connections. Not all hESC lines responded equally to SPIE; BG01V2 and BG03 formed large numbers of DA neurons, while BG02, ES02, and ES04 were much less responsive. SPIE did not induce DA differentiation of NE-4C, an embryonic mouse neuroectodermal cell line, P19, an mouse embryonic carcinoma cell line, or C17.2, a neonatal mouse cerebellar progenitor cell line. Therefore the application of SDF-1, PTN, IGF2, and EFNB1, to certain hESC lines results in rapid generation of DA neurons under chemically-defined conditions, free of xenogenic cells or material. Experiments on modifying SPIE to be applicable for a wider range of hESC lines are currently in progress. (Some of these data have previously been published; T. Vazin et al., PLoS ONE, 4(8):e6606, 2009). Research was supported by the IRPs of NIDA and NIA, NIH, DHHS.