We developed a mathematical model to simulate shear stress-dependent nitric oxide (NO) production and transport in a 3D microcirculatory network based on published data. The model consists of a 100 μm × 500 μm × 75 μm rectangular volume of tissue containing two arteriole-branching trees, and nine capillaries surrounding the vessels. Computed distributions for NO in blood, vascular walls, and surrounding tissue were affected by hematocrit (Hct) and wall shear stress (WSS) in the network. The model demonstrates that variations in the red blood cell (RBC) distribution and WSS in a branching network can have differential effects on computed NO concentrations due to NO consumption by RBCs and WSS-dependent changes in NO production. The model predicts heterogeneous distributions of WSS in the network. Vessel branches with unequal blood flow rates gave rise to a range of WSS values and therefore NO production rates. Despite increased NO production in a branch with higher blood flow and WSS, vascular wall NO was predicted to be lower due to greater NO consumption in blood, since the microvascular Hct increased with redistribution of RBCs at the vessel bifurcation. Within other regions, low WSS was combined with decreased NO consumption to enhance the NO concentration.