Crucial to their survival and function as reparative cells within all human tissues, perivascular-derived mesenchymal stromal cells (hMSCs) actively sense and respond to variations in extracellular matrix (ECM) composition and changes in the mechanics of their local microenvironment. However, the underlying mechanisms defining how such fates are temporally modulated remains poorly understood. We have recently studied the dynamics of hMSC adhesion, mechano-sensing and -transduction on substrates of different adhesive peptide nanospacing, representative of those within ECM proteins, using a range of real-time fluorescent location and FRET-based biosensors to probe this complex problem with high spatial and temporal resolution, over timeframes of 2 hours to 14 days. These studies have revealed that changes in lateral spacing of adhesion motifs from 30 to 60 nm affect the temporal recruitment and activation of critical focal adhesion proteins and mechanotransduction signalling pathway modulators (including RhoGTPases and YAP/TAZ), which ultimately leads to the observed bias in cell fate commitment through the regulation of well-known morphogenic determinants, including the RUNX2 and Wingless/Integrated (Wnt) pathway. Smaller nanospacings recruit more FAs, generate higher tension force per FA, and bias upregulation of the Rac 1/ myosin IIA / YAP-TAZ / RUNX 2 pathway, confirming that observed switches in osteo- versus adipo-genic fate commitment are the result of (early) differences in tension force at focal adhesions of a little as 5pN! We know, however, that in response to differences in matrix mechanics or ligand presentation, hMSCs produce a range of other regulatory molecules, besides proteins. Could these pathway modulators offer a solution to overcoming the impacts of ‘perturbed’ ECM microenvironments on hMSC behaviour and function to effect functional tissue repair? To address this question, we have recently identified differentially expressed microRNAs (miRs) produced by hMSCs in response to varying matrix stiffness and RhoA activity, or post activation in an injury site. Thereafter, using hydrogels and self-assembling nanoparticles, we have shown that modulating these mechanosensitive miRNAs (through targeted delivery) can overcome local ‘soft’ mechanical cues to drive commitment to stiffer tissues (eg, bone), or suppress the creation of ‘stiff’ fibrotic tissue within an injured ‘soft’ tissue. The outcomes of these recent studies provide new understanding of the complex mechanisms regulating mechanosensing, mechanotransduction and differentiation, but also novel strategies with which to manipulate mechano-driven cell fate and significantly impact tissue engineering and regenerative medicine applications that focus on their exogenous use or endogenous manipulation.