When an embryonic stem cell (ESC) differentiates, it must both silence the ESC self-renewal program as well as activate new tissue-specific programs. In the absence of DGCR8, a protein required for microRNA (miRNA) biogenesis, mouse ESCs are unable to silence the ESC self-renewal program during differentiation. Screening by reintroduction of all known miRNAs one at a time into Dgcr8 -/- ESCs in differentiation-inducing conditions enabled the identification of numerous miRNAs which silence the ESC self-renewal program. Expression levels of many of these miRNAs are induced during ESC differentiation. Of these miRNAs, most are expressed in specific cell types whereas a single family, the let-7 family, is broadly expressed across differentiated cell types. In various assays of ESC self-renewal, let-7 family miRNAs rescue the inability of Dgcr8 -/- ESCs to silence self-renewal. However, let-7 miRNAs failed to silence self-renewal in wild-type ESCs, suggesting that ESC-expressed miRNAs inhibit the capacity of let-7 to silence self-renewal. Indeed, introduction of the embryonic stem cell cycle regulating (ESCC) miRNAs blocked the capacity of let-7 to induce silencing of self-renewal in Dgcr8 -/- ESCs. mRNA profiling and bioinformatic analysis showed that let-7 and ESCC miRNAs function in part through opposite regulation of Myc transcription factors and Lin28. The opposing regulation of these factors contributes to a network, which reinforces the switch from a self-renewing to a differentiated cell state. These results suggested that additional screen positive miRNAs function in similar antagonistic networks with ESCC miRNAs. Indeed, introduction of the ESCC miRNAs prevented the additional screen positive miRNAs from silencing self-renewal in Dgcr8 -/- ESCs. mRNA profiling and bioinformatic analyses suggest that screen positive miRNAs and the ESCC miRNAs oppositely regulate multiple molecular pathways including the G1/S cell cycle transition. Inhibition of the G1/S transition in wild-type ESCs promotes loss of markers of ESC self-renewal. These findings suggest that miRNAs through destabilization of the ESC cell-cycle may promote loss of ESC self-renewal during differentiation. These studies show that different classes of miRNAs positioned in the context of complex biological networks function to either promote or antagonize ESC self-renewal.