This study probes the effects of ethanol on the molecular mechanisms regulating the differentiation of embryonic stem (ES) cells towards neuroectodermal state, which may be responsible for the abnormalities observed in fetal alcohol spectrum disorders (FASD). The effects of ethanol on the early phase of ES cell differentiation have not been well characterized. Here, we investigate the stage-specific action of ethanol during early embryogenesis by an integrated experimental and computational modeling approach. Our experimental system consists of mouse ES cells and directed differentiation to neuroectodermal fate in the presence of ethanol. Experimental single-cell multiplex data on the expression of the ES core transcription factors (TFs), Sox2, Oct4 and Nanog were obtained simultaneously by multicolor flow cytometry in live cells. Singlecell flow cytometric data were analyzed by ARACNE probabilistic modeling to construct transcriptional regulatory networks and quantify the TFs interactions in a pairwisemanner. Our analysis indicates that during differentiation towards neuroectodermal fate ethanol accelerates (i) the decline of the expression levels of Sox2 and Nanog, and (ii) the decreasing strength of the correlative interactions between the core TFs which is also reflected in (iii) an advanced differentiation phenotype.