Nanog, Sox2, and Oct4 are key transcription factors critical for the pluripotency and self-renewal of embryonic stem (ES) cells. Their downregulations lead to differentiation, accompanied with changes in cell motility. Whether these factors impact cell motility directly, however, is not clear. Here we addressed this question by initially assessing their effect in non-stem cells. We found that the ectopic expression of Nanog, Sox2, or Oct4 markedly inhibited ECV304 cell migration. Detailed examinations revealed that Nanog induced disorganizations of the actin cytoskeleton and peripheral localizations of focal adhesions. These effects required its DNA-binding domain and are thus transcription dependent. Furthermore, thymosin β4 and Rnd3 were identified as its downstream targets. Their depletions in ECV304 cells by RNAi phenocopied the ectopic expression of Nanog in both cell motility and actin organization, whereas their ectopic expressions rescued the migration defect of Nanog overexpression. Both proteins were upregulated during mouse ES cell differentiation. Their levels in the pluripotent mouse P19 cells also increased upon Nanog ablation, coincident with an increase in cell motility. Moreover, persistent expression of Nanog in zebrafish embryos suppressed gastrulation and cell migration. These results indeed suggest a dual role of certain transcription factors in the orchestration of differentiation and motility.