Previous studies have demonstrated that the physical properties of biomaterials can be adjusted to program therapeutically relevant functions in encapsulated mesenchymal stromal cells (MSCs). However, the intracellular pathways affected by mechanical cues in this cell type are not well understood. An understanding of the pertinent factors involved would not only aid in the rational design of substrates but also uncover targetable mechanisms that can facilitate the engineering of MSCs for cell therapies. Here, a bone marrow-mimetic hydrogel is employed to systematically explore the stiffness-responsive transcriptome of MSCs. High matrix rigidity impedes integrin-collagen adhesions which yields changes in cell morphology that are characterized by a contractile network of actin proximal to the cell membrane. This results in a suppression of ECM-regulatory genes involved in the remodeling of collagen fibrils and an upregulation of secreted immunomodulatory factors. Moreover, an investigation of long non-coding RNAs reveals that CYTOR contributes to these stiffness-driven changes in gene abundance. A robust knockdown of CYTOR using antisense oligonucleotides enhances the expression of numerous mechanoresponsive cytokines and chemokines to levels exceeding what is achievable by modulating matrix stiffness alone. Taken together, these findings emphasize the utility in studying previously unexplored mechanisms of mechanotransduction to inform novel strategies for enhancing the efficacy of MSC-based therapies. Overall design: To investigate the stiffness-sensitive transcriptome of MSCs and assess the role of intracellular contractility in mechanotransduction, we cultured human mesenchymal stromal cells (MSCs) in a stiffness-tunable hydrogel consisting of an interpenetrating network (IPN) of alginate and rat tail collagen I. The cells were encapsulated in IPNs with storage moduli of 150, 500, and 2000 Pa for 24 or 48 hours with either DMSO or blebbistatin treatment. We preformed RNA-sequencing on two cDNA libraries: poly(A)-enriched and ribosomal RNA-depleted. The poly(A)-enriched libraries were comprised of 36 samples aquired from three biological replicates of all permutations of stiffness, blebbistatin, and time conditions described above. The ribosomal RNA-depleted libraries were formed from a second set of 9 samples of MSCs cultured at the three different stiffnesses for 48 hours without any chemical treatment.