Transcriptional profiling is a powerful approach for studying mouse development, physiology and disease models. Here we describe a protocol for mouse thiouracil tagging (TU tagging), a transcriptome analysis technology that includes in vivo covalent labeling, purification and analysis of cell type-specific RNA. TU tagging enables the isolation of RNA from a given cell population of a complex tissue, avoiding transcriptional changes induced by cell isolation trauma, as well as the identification of actively transcribed RNAs and not preexisting transcripts. Therefore, in contrast to other cell-specific transcriptional profiling methods based on the purification of tagged ribosomes or nuclei, TU tagging provides a direct examination of transcriptional regulation. We describe how to (i) deliver 4-thiouracil to transgenic mice to thio-label cell lineage-specific transcripts, (ii) purify TU-tagged RNA and prepare libraries for Illumina sequencing and (iii) follow a straightforward bioinformatics workflow to identify cell type-enriched or differentially expressed genes. Tissue containing TU-tagged RNA can be obtained in 1 d, RNA-seq libraries can be generated within 2 d and, after sequencing, an initial bioinformatics analysis can be completed in 1 additional day.