The ability of mesenchymal stem cells to sense changes in extracellular matrix (ECM) composition in their local microenvironment is crucial to their survival. Transmembrane receptors that have specificity for peptide sequences within individual ECM molecules permit cells to rapidly assess the mechanical properties of the ECM, and respond by recruiting intracellular proteins to form focal adhesion (FA) complexes. The cell-adhesive domain arginine-glycine-aspartate (RGD) that is present in various ECM proteins has been extensively utilized to modify biomaterial surfaces and cell adhesion. Our previous data showed that increased lateral nanospacing (30 to 60 nm) of RGD peptides led to lower cell spread areas and less mature FA formation in human mesenchymal stem cells (hMSCs). However, the exact underlying mechanism driving these substantial differences remains unclear. In this work, we created tailored surfaces of self-assembled, azide-functionalized polystyrene-block-poly (ethylene oxide) copolymers (PS-PEO-N3) with controlled lateral spacing of RGD peptides (~30 and 60nm) on PEO-N3 nanodomains. Fluorescence resonance energy transfer (FRET)-based reporters were utilized in hMSCs to investigate the molecular effectors and regulators involved in mechanotransductive signaling, including vinculin, FAK, Src, RhoA and Rac 1. We observed that smaller (30nm) nanospacing resulted in lower vinculin tension sensor (VinTS) FRET activity, indicating the development of higher levels of tension at the site of focal adhesions compared to larger (60nm) nanodomain spacings. By combining fluorescence-lifetime imaging microscopy (FLIM) and VinTS FRET, we observed that the recruited vinculin also remained in these FAs for longer at smaller nanodomain spacings, with fluorescence life times of up to t=2.78ns, compared to t=2.26ns on larger nanodomain spacings. More FAK-GFP expression was found to colocalize at FA sites with longer FA lifetime on smaller nanodomain spacings when compared to larger nanospacings, suggesting the formation of more mature FAs. In terms of modulation of intracellular signaling pathways associated with mechanotransduction, higher RhoA and Src FRET activity and lower Rac FRET activity were noted when the lateral spacing of peptides was decreased from 60nm to 30nm. This study shows that smaller lateral nanospacing of adhesion peptides enables the recruitment of greater numbers FA proteins, enabling the transmission and development of higher levels of tension into the cytoskeleton, and the biased upregulation of critical mechanosensitive RhoGTPases. This investigation provides new mechanistic insights into how nanodomain lateral spacing can modify hMSCs behavior and correlate with changes in stem cell fate.