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T-Box Transcription Factor 18 is a member of the T-box family, encoding TBX18 protein. As a transcriptional repressor, it related to developmental processes of a majority of tissues and organs and plays crucial part in the embryonic development of sinoatrial node. Using an episomal vector-based CRISPR/Cas9 system, we have established a homozygous TBX18 knockout (TBX18-KO) human embryonic stem cell (hESC) line. This newly TBX18-/- hESC line display normal pluripotency, morphology, karyotype and trilineage differentiating capacity. This cell line may provide a powerful tool to investigate the role of TBX18 gene in sinoatrial node development in future.