This study investigates whether stem cells retain memory of past physical signals and how mechanical dosing influences their fate. Human mesenchymal stem cells (hMSCs) were cultured on soft poly(ethylene glycol) (PEG) hydrogels with different Young’s moduli (E ~ 2 kPa) and compared to stiff tissue culture polystyrene (TCPS; E ~ 3 GPa). The activation of YAP and TAZ, as well as the pre-osteogenic transcription factor RUNX2, was found to depend on prior culture time on stiff substrates. Mechanical dosing of hMSCs on initially stiff and then soft PEG hydrogels resulted in either reversible or irreversible activation of YAP/TAZ and RUNX2. Increased mechanical dosing on supraphysiologically stiff TCPS biased hMSCs toward osteogenic differentiation. The study concludes that stem cells possess mechanical memory, with YAP/TAZ acting as an intracellular mechanical rheostat that stores information from past physical environments and influences cell fate. The research highlights that stem cells respond to mechanical signals from the extracellular matrix (ECM), with recent studies clarifying the effects of modulus, adhesive ligand density, and nanotopography on cell fate. The study demonstrates that YAP/TAZ act as intracellular mechanical sensors, translating physical information into protein expression by localizing to the nucleus and regulating mRNA expression. The findings suggest that mechanical dosing can irreversibly influence stem cell fate, with YAP/TAZ playing a key role in this process. The study also shows that mechanical dosing on dynamic substrates can influence stem cell differentiation, with hMSCs cultured on stiff hydrogels showing increased osteogenic differentiation when transferred to soft hydrogels. The results indicate that mechanical dosing can bias stem cell differentiation toward osteogenesis, even in the presence of adipogenic signals. The study concludes that mechanical dosing and memory are important factors in stem cell fate, with implications for both fundamental understanding of cellular mechanotransduction and practical applications in stem cell culture and differentiation.This study investigates whether stem cells retain memory of past physical signals and how mechanical dosing influences their fate. Human mesenchymal stem cells (hMSCs) were cultured on soft poly(ethylene glycol) (PEG) hydrogels with different Young’s moduli (E ~ 2 kPa) and compared to stiff tissue culture polystyrene (TCPS; E ~ 3 GPa). The activation of YAP and TAZ, as well as the pre-osteogenic transcription factor RUNX2, was found to depend on prior culture time on stiff substrates. Mechanical dosing of hMSCs on initially stiff and then soft PEG hydrogels resulted in either reversible or irreversible activation of YAP/TAZ and RUNX2. Increased mechanical dosing on supraphysiologically stiff TCPS biased hMSCs toward osteogenic differentiation. The study concludes that stem cells possess mechanical memory, with YAP/TAZ acting as an intracellular mechanical rheostat that stores information from past physical environments and influences cell fate. The research highlights that stem cells respond to mechanical signals from the extracellular matrix (ECM), with recent studies clarifying the effects of modulus, adhesive ligand density, and nanotopography on cell fate. The study demonstrates that YAP/TAZ act as intracellular mechanical sensors, translating physical information into protein expression by localizing to the nucleus and regulating mRNA expression. The findings suggest that mechanical dosing can irreversibly influence stem cell fate, with YAP/TAZ playing a key role in this process. The study also shows that mechanical dosing on dynamic substrates can influence stem cell differentiation, with hMSCs cultured on stiff hydrogels showing increased osteogenic differentiation when transferred to soft hydrogels. The results indicate that mechanical dosing can bias stem cell differentiation toward osteogenesis, even in the presence of adipogenic signals. The study concludes that mechanical dosing and memory are important factors in stem cell fate, with implications for both fundamental understanding of cellular mechanotransduction and practical applications in stem cell culture and differentiation.