The Embryonic Stem cell Test (EST) is an in vitro alternative test designed for the prediction of embryotoxicity. The endpoint of the test is the interference with mesoderm-derived cardiac muscle differentiation observed under the microscope as beating muscle foci. The relative subjectivity of this endpoint, as well as the applicability domain and related predictivity need further to be defined to facilitate implementation of the EST into regulatory strategies. The use of transcriptomics techniques to monitor differentiation-related gene expression changes in the EST might improve the EST in each of these aspects. Therefore, we studied the gene expression profile in embryonic stem cells (ESC) in the early phase of differentiation and its modulation by exposure to the well known embryotoxicant monobutyl phthalate (MBP). Cells were exposed from the early embryoid body stage onwards and RNA was collected after 6, 12 and 24h of exposure. Samples were hybridized to spotted microarrays, containing 21,997-mer oligonucleotides. Differential gene expression patterns were analyzed. A total number of 43 genes that were found to be upregulated in this study as a consequence of induction of cardiomyocyte differentiation were combined in a gene set, named 'VAN_DARTEL_HEARTDIFF_24H'. Gene Set Enrichment Analysis (GSEA) comparative analysis using multiple gene set collections clearly showed that temporal changes in gene expression were functionally related to cardiomyocyte differentiation. Furthermore, exposure of embryoid bodies (EB) to MBP increased expression of pluripotency-, proliferation- and nonmesodermal differentiation-related gene sets, which indicates inhibition of mesodermal differentiation. The inhibition of mesoderm-derived cardiomyocyte differentiation by MBP exposure was most obvious through the downregulation of our novel gene set identified in this study, 'VAN_DARTEL_HEARTDIFF_24H', which specifically describes the niche of early cardiomyocyte differentiation. The gene set defined in this study might serve as a starting point for defining a dedicated gene set for early detection of embryotoxicity in the EST. Such a gene set may serve as an improved endpoint in the EST as compared to morphology, and will allow a more detailed definition of the applicability domain and predictivity of EST.