Precise control of gene expression is of paramount importance for proper embryonic development. Although a number of microRNAs (miRNAs) has been implicated in fine-tuning mRNA translation during development, their exact roles for gastrulation, particularly in connection with functional targets, have yet to be clarified, with regard to stage-specific cell migration to form three embryonic germ layers. We found that miR-124a is expressed in human embryonic stem cells (hESC), but is gradually downregulated during embryoid body (EB) formation in vitro. We also provide evidence that SLUG and IQGAP1, which modulates rearrangement of the migratory cytoskeleton, are specific targets for miR-124a during EB formation. Furthermore, we show that the beginning of cell migration, a hallmark event in gastrulation, is tightly coupled with downregulation of miR-124a during EB formation and induction of SLUG and IQGAP1. Overexpressed miR-124a in hESC reduced expression of SLUG and IQGAP1 and blocked migratory cell behavior in EB. An expression level of MIXL1, associated with gastulation process, was also inversely correlated with expression of miR-124a. Taken together, our results strongly suggest that miR-124a may play an active role in inhibiting hESCs from differentiation into EB by downregulating expression of SLUG and IQGAP1, thereby maintaining stemness. STEM CELLS 2010; 28:1550–1559.