Functional analysis of multiple genes is key to understanding gene regulatory networks controlling embryonic development. We have developed an integrated vector system for inducible gene silencing by shRNAmir-mediated RNA interference in mouse embryos, as a fast method for dissecting mammalian gene function. For validation of the vector system, we generated mutant phenotypes for Brachyury, Foxa2 and Noto, transcription factors which play pivotal roles in embryonic development. Using a series of Brachyury shRNAmir vectors of various strengths we generated hypomorphic and loss of function phenotypes allowing the identification of Brachyury target genes involved in trunk development. We also demonstrate temporal control of gene silencing, thus bypassing early embryonic lethality. Importantly, off-target effects of shRNAmir expression were not detectable. Taken together, the system allows the dissection of gene function at unprecedented detail and speed, and provides tight control of the genetic background minimizing intrinsic variation.