Research in the mouse embryo and into mouse embryonic stem (ES) cells in culture has identified several regulators of primitive streak intermediate (PSI) formation. Although the involvement of multiple growth factors and signalling pathways has been demonstrated, translation of this information into a commercial paradigm has been limited by the expense of using recombinant proteins in large-scale applications and/or the requirement for genetic manipulation of cells to facilitate cell purification from heterogeneous populations. Small molecule regulators of signalling pathways have been hypothesized as agents that will allow greater control of differentiation in ES cells in vitro. Using mouse early primitive ectoderm-like (EPL) cells as a model for epiblast-like human ES cellsand a high throughput screening assay (HTSA), we have identified two small molecules (C4 and F10) that induce the expression of Brachyury (T) in our system. When added to differentiating EPL cells C4 and F10 induced formation of cells expressing PSI markers, including Brachyury, Fgf8 and Mixl1. T-expressing cells induced in response to either chemical showed reduced expression of E-cadherin, suggesting that the responding cells were undergoing an epithelial-to-mesenchyme transition (EMT). Further differentiation of cell populations treated with C4 or F10 formed fewer neurons when compared to controls. All of these features are consistent with the formation of PSIs in response to the chemicals. The action of C4 and F10 was specific to differentiating EPL cells as neither chemical induced differentiation in mouse ES cells. Biochemically, the action of the chemicals required active ERK signalling. Preliminary evidence, however, suggests that the chemicals interact with distinct targets to effect cell differentiation. Lastly, C4 was shown to induce T expression in human ES cells; this shows that a HTSA based on EPL cells can identify compounds capable of modulating human ES cell differentiation.