Atomic force microscopy (AFM) has emerged as a promising tool to characterize the mechanical properties of biological materials and cells. In our studies, undifferentiated and early differentiating mouse embryonic stem cells (mESCs) were assessed individually using an AFM system to determine if we could detect changes in their mechanical properties by surface probing. Probes with pyramidal and spherical tips were assessed, as were different analytical models for evaluating the data. The combination of AFM probing with a spherical tip and analysis using the Hertz model provided the best fit to the experimental data obtained and thus provided the best approximation of the elastic modulus. Our results showed that after only 6 days of differentiation, individual cell stiffness increased significantly with early differentiating mESCs having an elastic modulus two- to threefold higher than undifferentiated mESCs, regardless of cell line (R1 or D3 mESCs) or treatment. Single-touch (indentation) probing of individual cells is minimally invasive compared to other techniques. Therefore, this method of mechanical phenotyping should prove to be a valuable tool in the development of improved methods of identification and targeted cellular differentiation of embryonic, adult, and induced-pluripotent stem cells for therapeutic and diagnostic purposes.