The blood brain barrier (BBB) plays a significant role in maintaining homeostasis within the brain by regulating transport in and out of the brain microvasculature. Cell-cell junctions are an integral component that aid in the selective permeability of the BBB. Both mechanical and biochemical cues help regulate the cell-cell junctions. In particular, matrix stiffness is one of the mechanical cues that has been previously shown to impact the cell-cell junctions of brain microvascular endothelial cells (BMECs), and we hypothesized that this may occur through changes in cell stiffness. To understand the impact of matrix stiffness on the cell stiffness of induced pluripotent brain microvascular endothelial cells (iBMECs), atomic force microscopy (AFM) was conducted on the iBMECs which were plated on extracellular matrix coated hydrogels of varying stiffnesses ranging from 1 to 194 kPa. The role of astrocytes and pericytes, which are critical accompanying cells for BBB function, in regulating the cell stiffness was also investigated. Without the introduction of the astrocytes and pericytes, the iBMECs were most stiff on the 2.5 kPa hydrogels, and the Young’s modulus was highest in the tricellular junction region. The introduction of the astrocytes and pericytes resulted in a slight decline in iBMEC stiffness, although the tricellular junctions remained the stiffest regions in the monolayer. In future studies, the impact of metastatic breast cancer cells on cell stiffness will also be investigated, towards understanding how matrix stiffness impacts the BBB mechanical properties and barrier function during tumor cell metastasis.