The relative ease of mouse Embryonic Stem Cells (mESCs) culture and the potential of these cells to differentiate into any of the three primary germ layers: ectoderm, endoderm and mesoderm (pluripotency), makes them an ideal and frequently used ex vivo system to dissect how gene expression changes impact cell state and differentiation. These efforts are further supported by the large number of constitutive and inducible mESC mutants established with the aim of assessing the contributions of different pathways and genes to cell homeostasis and gene regulation. Gene product abundance is controlled by the modulation of the rates of RNA synthesis, processing, and degradation. The ability to determine the relative contribution of these different RNA metabolic rates to gene expression control using standard RNA-sequencing approaches, which only capture steady state abundance of transcripts, is limited. In contrast, metabolic labeling of RNA with 4-thiouridine (4sU) coupled with RNA-sequencing, allows simultaneous and reproducible inference of transcriptome wide synthesis, processing, and degradation rates. Here we describe, a detailed protocol for 4sU metabolic labeling in mESCs that requires short 4sU labeling times at low concentration and minimally impacts cellular homeostasis. This approach presents a versatile method for in-depth characterization of the gene regulatory strategies governing gene steady state abundance in mESC.