ABSTRACT MicroRNAs (miRNAs) have been shown to be important in early development and maintenance of human embryonic stem cells (hESCs). The miRNA miR‐302–367 is specifically expressed in hESCs, and its expression decays on differentiation. We previously identified the structure of the gene coding for the human miR‐302–367 cluster and characterized its promoter. The promoter activity was functionally validated in hESCs, opening up new avenues to further investigate how these miRNA molecules fit in the complex molecular network conferring “sternness” properties to hESCs. The physiological roles of specific miRNA‐mRNA interactions remain largely unknown. Here, we investigated putative miR‐302–367 mRNA targets in hESCs, potentially relevant for ESC biology. We found that the Nodal inhibitors Lefty1 and Lefty2 are post‐transcriptionally targeted by miR‐302s in hESCs. Functional analyses indicate that miR‐302s negatively modulate the level of lefties, and become upstream regulators of the TGFβ/Nodal pathway, functioning via Smad‐2/3 signaling. Overexpression of the miR‐302–367 cluster in hESCs causes a delay in early hESC differentiation, as measured by enhanced levels of ESC‐specific transcription factors, coupled to a faster teratoma formation in mice transplanted with miR‐302–367‐expressing hESCs and a concomitant impairment of germ layer specification, displaying robust decreased levels of early mesodermal, endodermal, and ectoder‐mal specific markers. These findings suggest that Lefty is negatively modulated by miR‐302s in hESCs, which plays an important role in maintaining the balance between pluripotency and germ layer specification.—Barroso‐delJesus, A., Lucena‐Aguilar, G., Sanchez, L., Ligero, G., Gutierrez‐Aranda, I., Menendez, P. The Nodal inhibitor Lefty is negatively modulated by the microRNA miR‐302 in human embryonic stem cells. FASEB J. 25, 1497–1508 (2011). www.fasebj.org