RNA interference (RNAi) is an efficient method for silencing genes in cultured cells. Here we describe a simple RNAi approach for silencing genes in a cell type–specific and tissue-specific way in vivo. The approach, which mimics the means by which naturally occurring 'microRNA's are generated, uses a tissue-specific polymerase II promoter to drive the expression of a short hairpin RNA (shRNA) directed against the gene target. The shRNA is cleaved by ubiquitously expressed endonucleases to form an active small interfering RNA of about 22 nt. As a proof of principle, it has been shown that expression of a shRNA directed against the transcription factor Wilms tumor 1 in transgenic mice reduces that protein specifically in nurse cells in the testis. Our transgenic RNAi approach offers a cost-effective means of rapidly (within months) addressing the function(s) of genes of interest in a wide variety of specific cell types and tissues in mice in vivo.