Potassium niobate nonlinear nanoparticles are used for the first time to monitor the evolution of embryonic stem cells (ESC) by second harmonic microscopy. These particles feature the complete absence of photo‐bleaching and unlimited excitation wavelength flexibility. The potential of this approach is made evident for tissue‐regeneration studies and applications, by capturing a high‐speed movie of ESC‐derived cardiomyocytes autonomously beating within a cluster. Time‐resolved data are analyzed to retrieve 3D information of the contraction pattern at the cellular level. Harmonic nanoparticles, thanks to the absence of bleaching and blinking, together with the possibility to be excited at any wavelength, are perfectly suited for long‐term tracking and depth tissue imaging of differentiating stem cell structures with straightforward applications in regenerative medicine. Their potential is made evident by capturing and analyzing a high‐speed movie of embryonic‐stem‐cell‐derived cardiomyocytes autonomously beating.