The frequent detection of tetrabromobisphenol A (TBBPA) in the human body, especially in umbilical cord serum and breast milk, has raised concerns about TBBPA potential effects on embryonic development. The differentiation of embryonic stem cells (ESCs) in vitro can serve as a model for the early stages of embryonic development. In this study, we differentiated mouse ESCs via 3D aggregates called embryoid bodies in presence of environment and human relevant TBPPA concentrations for 28 days. We collected samples at different time points and analyzed TBBPA-dependent global gene expression changes by RNA-seq. Our analyses revealed a potential TBBPA multifaceted developmental toxicity with effects on the nervous and cardiac/skeletal muscle systems. Mechanistically, our findings suggest TBBPA endocrine disrupting activities in part via prolactin naling.