F-53B and PFOS are two per- and polyfluoroalkyl substances (PFASs) widely utilized in the metal plating industry as mist suppressants. Recent epidemiological studies have linked PFASs to cardiovascular diseases and alterations in heart geometry. However, we still have limited understanding of the effects of F-53B and PFOS on the developing heart. In this study, we employed a human embryonic stem cell (hESC)-based cardiac differentiation system and whole transcriptomics analyses to evaluate the potential developmental cardiac toxicity of F-53B and PFOS. We utilized F-53B and PFOS concentrations of 0.1-60 μM, covering the levels detected in human blood samples. We demonstrated that both F-53B and PFOS inhibited cardiac differentiation and promoted epicardial specification via upregulation of the WNT signaling pathway. Most importantly, the effects of F-53B were more robust than those of PFOS. This was because F-53B treatment disrupted the expression of more genes and led to lower cardiac differentiation efficiency. These findings imply that F-53B may not be a safe replacement for PFOS.