During migration in vivo, cells must pass through confined spaces, which requires extensive deformation of the cell body and nucleus, and can lead to substantial changes in chromatin organization. Previous work from our laboratory shows that confined migration through three-dimensional (3D) environments induces heterochromatin formation and decreases intergenic chromatin accessibility. However, the mechanisms of these changes in chromatin organization, including the role of specific histone-modifying enzymes and mechano-sensitive ion channels, remain unclear. Calcium signaling and the role of mechanosensitive channels during 3D confined migration also have yet to be characterized. Using different collagen and custom-made polydimethylsiloxane (PDMS) based microfluidic devices that mimic interstitial spaces in vivo, I demonstrate that confined migration induced heterochromatin (CMiH) occurs during migration through a range of 3D environments and is more subtle than initially reported. I show that CMiH is dependent on the activation of the histone modifying enzyme HDAC3 and other class I HDACs. I also show that mechanosensitive channels, including Piezo1, contribute to CMiH, but they do not influence migration speed. Furthermore, chelation of intracellular and extracellular calcium also contributes to CMiH in some contexts. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on cells migrating in novel PDMS migration devices reveals that confined migration alters chromatin accessibility at genes associated with diverse cellular functions. Taken together, I demonstrate that chromatin accessibility changes can depend more on whether migration takes place in 2D or 3D than initially reported. Using time-lapse experiments with cells expressing the calcium reporter GcaMP6, I also show that frequent, transient calcium flashes occur during confined migration, but that inhibition of mechanosensitive channel activity alone does not significantly alter calcium dynamics. Thus, this thesis provides additional insights into the mechanisms and functional consequences of migration-induced changes in chromatin organization and begins to characterize the role of calcium during confined migration.