Embryonic and induced pluripotent stem cells (ESCs and iPSCs) hold great promise for regenerative medicine. The therapeutic application of these cells requires an understanding of the molecular networks that regulate pluripotency, differentiation, and de-differentiation. Along with signaling pathways, transcription factors, and epigenetic regulators, microRNAs (miRNAs) are emerging as important regulators in the establishment and maintenance of pluripotency. These tiny RNAs control proliferation, survival, the cell cycle, and the pluripotency program of ESCs. In addition, they serve as barriers or factors to overcome barriers during the reprogramming process. Systematic screening for novel miRNAs that regulate the establishment and maintenance of pluripotent stem cells and further mechanistic investigations will not only shed new light on the biology of ESCs and iPSCs, but also help develop safe and efficient technologies to manipulate cell fate for regenerative medicine.