To develop a differentiation screening method using 3D culture with ES cells, EBs were cultured for 21 days, in a culture media of collagen gel and chemicals affecting differentiation. The chemicals consisted of cytokines, EGF, FGF, IL-6, IL-12, TNF-α and mLIF, vitamins such as L-ascorbic acid, cholecalciferol and folic acid, and BMP, Emdogain^R, four adrenocortical hormones such as Dexamethasone, Betamethasone, Betamethasone Valerate and Betamethasone Dipropionate. The results demonstrated that the myocardial pulsation rate decreased with IL-6, TNF-α, L-ascorbic acid or cholecalciferol, but there was no significant difference observed with other chemicals. Regarding the amount of osteocalcin, FGF induced 360% of that in the control, BMP induced 310% in the control and Cholecalciferol induced 180% that of that in the control. For other combinations, there were no significant differences observed. XPS also showed a tendency toward similar results. The results of this study have shown that the effects of chemical substances on ES cell differentiation can be determined with the 3D culture method by collagen.