<p>Myocardial infarction is associated with massive cardiomyocyte loss, and embryonic stem cells (ESCs), owing to their cardiomyocyte differentiation ability, have emerged as a promising therapy. One potential factor involved in regulating ESC-to-cardiomyocyte differentiation is activating transcription factor 3 (Atf3), and this study clarifies its involvement. Atf3 was knocked out (ko) in mouse ESCs (mESCs) with CRISPR/Cas9. Both wild-type (WT) and Atf3-KO mESCs formed embryoid bodies (EBs) over 6 days, followed by adherent culture to induce cardiomyocyte differentiation from days 7–12; there, Atf3-KO had more beating EBs, at higher frequencies, from Day 8 of differentiation than WT. They also had increased expression of mesodermal markers on Days 3–6 (ex. T), cardiac progenitor markers on Days 6–9 (ex. Pdgfra), and cardiomyocyte differentiation marker on Days 9–12 (ex. cardiac troponin T), as measured by RT-qPCR and flow cytometry. Furthermore, cardiomyocyte differentiation-associated differentially-expressed genes were significantly upregulated in Atf3-KO EBs, compared to WT, under Gene Set Enrichment Analysis of RNA sequencing. All these effects in Atf3-KO mESCs were reversed upon “rescue”, where Atf3 expression was restored in these cells. Therefore, Atf3-KO in mESCs promotes differentiation into mesodermal lineages, which further differentiate into cardiac progenitors, serving as a target for cell-based cardiac regeneration therapies.</p>