Objective ·To establish a T-box transcription factor Brachyury(T gene) fluorescence reporter cell line, in which foot-and-mouth disease virus 2A (F2A) and enhanced green fluorescent protein (EGFP) were knocked in at the end of mouse T gene (mT-F2A-EGFP) in mouse embryonic stem cells (mESCs) by CRISPR/Cas9n (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nickase)-mediated homologous-directed repair (HDR) technology. Methods ·First of all, the specific single guide RNA (sgRNA) plasmid targeting the sequence near the stop codon of the mouse T gene and the plasmid donor containing F2A-EGFP were constructed. These two plasmids were co-delivered into mESCs E14Tg2a (E14) by electroporation. In this way, the desired fluorescent marker EGFP with self-cleaving peptide F2A were introduced into the end of T gene via HDR. Then, the monoclonal cells, obtained after drug selection and verified by sequencing, were induced for differentiation as embryonic bodies (EB), of which the fluorescence signals of mT-F2A-EGFP were monitored by fluorescence microscope and flow cytometry. These reporter clones were also selected before and after differentiation by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), which detected the transcription levels of marker genes determing pluripotency, mesendoderm differentiation or ectoderm differentiation. In addition, the cell cycle and growth curve of these clones were detected. Meanwhile, alkaline phosphatase (AP) staining was used to detect the stem cell characteristics of these candidate clones. Finally, the clone T1 carrying mT-F2A-EGFP was selected for EB differentiation. Flow cytometry was used to sort out EGFP expression cells (EGFP^＋) and non-EGFP expressing cells (EGFP^－) from the EBs comprising multiple lineage cells upon differentiation, of which cell lineage markers were checked by RT-qPCR. Results ·EGFP was correctly inserted after the T gene in E14, whose fluorescence intensity reflected the expression level of endogenous T without observed side effects. When the fluorescence reporter clone T1 was differentiated, the EGFP⁺ cells sorted by flow cytometry mainly expressed mesendoderm marker genes. Conclusion ·The establishment of mESC line carrying mT-F2A-EGFP can realize rapid monitoring of the degree of T regulation, and track mesendoderm cells expressing T marker, EGFP in real time during differentiation.