Nuclear receptors and coregulators orchestrate diverse aspects of biological functions and inappropriate expression of these factors often associates with human diseases. The present study describes a conditional overexpression system consisting of a minigene located at the Rosa26 locus in the genome of mouse embryonic stem (ES) cells. Before activation, the minigene is silent due to a floxed STOP cassette inserted between the promoter and the transgene. Upon cre-mediated excision of the STOP cassette, the minigene constitutively expresses the tagged transgene driven by the ubiquitous CAGGS promoter. Thus, this system can be used to express target gene in any tissue in a spatial and/or temporal manner if respective cre mouse lines are available. Serving as proof of principle, the CAG-S-hCOUP-TFI allele was generated in ES cells and subsequently in mice. This allele was capable of conditionally overexpressing human chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) in all tissues tested upon activation by cre drivers. This allele was further subjected to address functionality of expressed COUP-TFI and the functional similarity between COUP-TFI and COUP-TFII. Expression of COUP-TFI in COUP-TFII-ablated uterus suppressed aberrant estrogen receptor-α activities and rescued implantation and decidualization defects of COUP-TFII mutants, suggesting that COUP-TFI and COUP-TFII are able to functionally compensate for each other in the uterus. A toolbox currently under construction will contain ES cell lines for overexpressing all 48 nuclear receptors and selected 10 coregulators. Upon completion, it will be a very valuable resource for the scientific community. Several ES cells are currently available for distribution.