AbstractEmbryonic stem (ES) cells differentiate towards all three germ layers, including cardiac cells and leukocytes, and may be therefore suitable to model inflammatory reactions in vitro. In the present study, embryoid bodies differentiated from mouse ES cells were treated with increasing doses of lipopolysaccharide (LPS) to mimic infection with gram‐negative bacteria. LPS treatment dose‐dependent increased contraction frequency of cardiac cell areas and calcium spikes and increased protein expression of α‐actinin. LPS treatment increased the expression of the macrophage marker CD68 and CD69, which is upregulated after activation on T cells, B cells and NK cells. LPS dose‐dependent increased protein expression of toll‐like receptor 4 (TLR4). Moreover, upregulation of NLR family pyrin domain containing 3 (NLRP3), IL‐1ß and cleaved caspase 1 was observed, indicating activation of inflammasome. In parallel, generation of reactive oxygen species (ROS), nitric oxide (NO), and expression of NOX1, NOX2, NOX4 and eNOS occurred. ROS generation, NOX2 expression and NO generation were downregulated by the TLR4 receptor antagonist TAK‐242 which abolished the LPS‐induced positive chronotropic effect of LPS. In conclusion, our data demonstrate that LPS induced a pro‐inflammatory cellular immune response in tissues derived from ES cells, recommending the in vitro model of embryoid bodies for inflammation research.