This paper focuses on the design of a disturbance rejection controller for a tailless aircraft based on the technique of nonlinear dynamic inversion (NDI). The tailless aircraft model mounted on a three degree-of-freedom (3-DOF) dynamic rig in the wind tunnel is modeled as a nonlinear affine system subject to mismatched disturbances. First of all, a baseline NDI attitude controller is designed for sufficient stability and good reference tracking performance of the nominal system. Then, a nonlinear disturbance observer (NDO) is supplemented to the baseline NDI controller to estimate the lumped disturbances for compensation, including unmodeled dynamics, parameter uncertainties, and external disturbances. Mathematical analysis demonstrates the convergence of the employed NDO and the resulting closed-loop system. Furthermore, an anti-windup modification is applied to the NDO for control performance preserving in the presence of actuator saturation. Subsequently, the designed control schemes are preliminarily validated and compared via simulations. The baseline NDI controller demonstrates satisfactory attitude tracking performance in the case of nominal simulation; the NDO augmented NDI controller presents significantly improved ability of disturbance rejection when compared with the baseline NDI controller in the case of robust simulation; the anti-windup modified scheme, rather than the baseline NDI controller nor the NDO augmented NDI controller, can preserve the closed-loop performance in the case of actuator saturation. Finally, the baseline NDI scheme and the NDO augmented NDI scheme are implemented and further validated in the wind tunnel flight tests, which demonstrate that the experimental results are in good agreement with that of the simulations.