Immune cells in tissues often face various physical barriers imposing spatial confinement due to migration through cell-cell junctions and extracellular matrix architecture. In these contexts, the nucleus of the cell experiences severe deformation, and constitutes a rate-limiting step when cells migrate though confined spaces. The nucleus plays a central role in determining how cells respond to spatial confinement. While this has been studied in other cell types including endothelial cells, cancer cells, and others, immune cells are extremely understudied, even though they contribute to many essential physiological processes and disease pathologies. Here, we focus on macrophages, which are innate immune cells that contribute to numerous pathologies including cancer. I designed an agarose-based confinement device to induce precisely controlled heights of cellular deformation, mimicking spatial confinement. I then applied this device to improve our understanding of how macrophages respond to spatial confinement. Using transcriptomic approaches, my work revealed that macrophages rapidly upregulate many transcription factors and biological processes in response to various degrees of spatial confinement. I also identified that pro-inflammatory polarization of macrophages causes nuclear stiffening in the absence of lamins, key proteins previously thought to universally regulate nuclear stiffness. This thesis thereby expands upon our knowledge of nuclear mechanobiology in macrophages.