As such they provide an in vitro model for development and a system that possesses considerable therapeutic potential. Recently, several systems have been developed that provide an insight into the roles that miRNAs play in ES cells by knocking out components of the miRNA processing pathway. Depletion of both DICER1 and DGCR8 proteins in mouse ES cells perturbs the cell cycle leading to an accumulation of cells in the G1 phase. These mutant ES cells are also unable to complete differentiation. By studying systems such as these it has become apparent that one of the most 4-(Benzyloxy)phenol highly expressed mouse ES cell miRNA clusters plays a fundamental role in the regulation of the mouse ES cell cell-cycle and differentiation. Many of the miRNAs within this cluster and other miRNAs that are highly expressed in mouse ES cells, share a high degree of sequence identity within their seed region and are consequently expected to share target mRNAs. Indeed these miRNAs have demonstrated a degree of functional redundancy in their regulation of the embryonic stem cell cycle. We describe a mouse ES cell line depleted in the expression of Dgcr8 and canonically processed miRNAs. This allows us to reintroduce miRNAs into a system with limited miRNA functional redundancy so targets should no longer be saturated by endogenous miRNA expression. Through a simple system by which miRNAs are reintroduced individually to these cells and subsequent mRNA expression changes are measured by microarray, we were able to partially rescue the wild-type ES cell mRNA expression profile and identify lists of mRNA transcripts that are likely targets of a number of miRNAs within wild type ES cells. In this way we are able to propose functions for individual miRNAs, uncover a broad network of the targets of miRNAs in ES cells and identify both basal transcription factors and the mediator complex as global/shared routes by which ES cell miRNAs appear to converge to regulate a wider cohort of secondary targets within these cells. A system depleted of the vast majority of miRNAs provides an opportunity for the identification of miRNA targets in a clean background. The targets of individual miRNAs will no longer be saturated by endogenously expressed miRNAs allowing a more thorough investigation of target interactions by miRNA transfection assays. Furthermore, such experiments will not encounter problems associated with functional redundancy of related miRNAs that may impede miRNA knockout and knockdown assays. In this study we have presented a comprehensive experimental approach to miRNA target detection in ES cells. The generation of a Dgcr8-deficient cell line provides an excellent system for the reintroduction of miRNAs for target identification against a “clean” background. As a consequence of developing this system, we have also demonstrated that miR-1186 is potentially processed in a Dgcr8-independent manner. Recently miRBase has begun to make its criteria for miRNA annotation more stringent. This should allow more accurate annotation of miRNAs based on RNA-seq data. We have demonstrated that systems such as that presented here can be used to interrogate miRBase to Albaspidin-AA shortlist miRNA annotations worthy of further scrutiny. Indeed, similar efforts have begun, testing the maturation of overexpressed miRNA hairpins in the context of dominant-negative alleles of Drosha or Dicer. As we have noted, a number of small RNAs that appeared to be processed in a Dgcr8-independent manner have recently been removed from miRBase. These small RNAs all appear to overlap either ribosomal or tRNA genes. While it is clear that the correct classification of small RNA fragments can be a complicated process, small RNAs of disparate origin may possess miRNAlike function. As such our work clearly highlights a current and multifarious issue facing the field of miRNA biology.