Fibronectin is an important protein that is able to bind to other fibronectin molecules and to cell surface receptors. In doing so, the interactions fibronectin can perform is important for the processes of cell migration and tissue formation. Understanding the properties of fibronectin and fibril assembly is useful for areas such as wound healing, where fibronectin molecules are assembled to protect the tissue and to perform other tasks. Because of these reasons, it is important to understand how fibronectin is assembled and how its properties affect the fibril assembly, which in return affects the way the cell matrix operates. Previously published papers illustrate that the properties of fibronectin affect the mechanotransduction process, the cell conversion of mechanical stimulus to chemical, and this leads to various changes of the fibril assembly. However, the question that now comes to focus is what variables affect the fibril assembly? The two main variables that come into question is the substrate stiffness (ksub) (pN/nm) and the actin velocity (Vu) (nm/s). In order to test this hypothesis, several fibril assembly simulations were performed via MATLAB based upon the Weinberg-Mair-Lemmon Fibronectin Model. These simulations were performed by varying the parameters of substrate stiffness and actin velocity as well as fibril size, which affect the various measurements of the fibronectin, such as stretched length, relaxed length, etc. Through these various experiments, it was determined that the actin velocity and fibril size had the greatest impacts in affecting the fibronectin’s properties and its assembly.