A fluidic environment affects mechanochemical characteristics of embryonic stem cells (ESCs). Perfusion is recognized as an attractive culture mode of ESCs since the steady fluidic state can enhance ESCs’ controllability, supporting a unique cell culture condition. Cellular membrane motility presents important information about cellular dynamics such as adhesion, spreading, and migration. Thus, an investigation of the perfusion-induced membrane motility is significant to understand the mechanochemical behavior of ESCs in the steady culture state. In this research, we suggest Lfr, the ratio of circumferential membrane unattached to other cells’ to the cell’s circumference, as a new parameter to characterize cells’ shape and motility. Lfr of embryonic stem cells has positive correlations with cellular area (Ar) and free peripheral length (Lf) but a negative correlation with roundness (Rn). We also propose a mathematical model representing ESCs’ membrane motilities and demonstrate their colonical behavior.