A crystal plasticity finite-element model, which explicitly and directly represents the complex microstructures of a non-metallic agglomerate inclusion within polycrystal nickel alloy, has been developed to study the mechanistic basis of fatigue crack nucleation. The methodology is to use the crystal plasticity model in conjunction with direct measurement at the microscale using high (angular) resolution-electron backscatter diffraction (HR-EBSD) and high (spatial) resolution-digital image correlation (HR-DIC) strain measurement techniques. Experimentally, this sample has been subjected to heat treatment leading to the establishment of residual (elastic) strains local to the agglomerate and subsequently loaded under conditions of low cyclic fatigue. The full thermal and mechanical loading history was reproduced within the model. HR-EBSD and HR-DIC elastic and total strain measurements demonstrate qualitative and quantitative agreement with crystal plasticity results. Crack nucleation by interfacial decohesion at the nickel matrix/agglomerate inclusion boundaries is observed experimentally, and systematic modelling studies enable the mechanistic basis of the nucleation to be established. A number of fatigue crack nucleation indicators are also assessed against the experimental results. Decohesion was found to be driven by interface tensile normal stress alone, and the interfacial strength was determined to be in the range of 1270–1480 MPa.