Purpose <p>Pluripotent stem cells (PSCs) can spontaneously differentiate, maintaining an undifferentiated state is crucial for regenerative medicine using PSCs. As they are inherently unstable and gradually lose their undifferentiated state, even under routine culture conditions. This “collapse” of the undifferentiated state may be distinct from directed differentiation, which is generally studied and remains poorly characterized at the molecular level. </p> Methods <p>In this study, we mimicked spontaneous differentiation and modeled the collapse of the undifferentiated state by removing mouse embryonic fibroblasts. Next, we analyzed global gene expression alterations using DNA microarrays. </p> Results <p>We demonstrated that the tendency of undifferentiated states to collapse varied by cell line and that spontaneous differentiation differed from semi-normal differentiation by embryoid body formation. We also identified several collapse-specific genes, including <i>B-cell CLL/lymphoma 6 member B</i>, <i>Forkhead box N4</i>, <i>KIT proto-oncogene</i>, <i>Tet methylcytosine dioxygenase 2</i>, <i>fibroblast growth factor receptor 1</i>, <i>fibroblast growth factor receptor</i> and <i>distal-less homeobox 4</i>. </p> Conclusion <p>These collapse-specific genes are expected to contribute to the elucidation of the mechanism of differentiation collapse and become useful markers in the cell population production process in regenerative medicine.</p>