Understanding the deformation behavior of metallic materials containing nanotwins (NTs), which can enhance both strength and ductility, is useful for tailoring microstructures at the micro- and nano- scale to enhance mechanical properties. Here, we construct a clear deformation pattern of NTs in austenitic stainless steel by combining in situ tensile tests with a dislocation-based theoretical model and molecular dynamics simulations. Deformation NTs are observed in situ using a transmission electron microscope in different sample regions containing NTs with twin-lamella-spacing (λ) varying from a few nanometers to hundreds of nanometers. Two deformation transitions are found experimentally: from coactivated twinning/detwinning (λ < 5 nm) to secondary twinning (5 nm < λ < 129 nm), and then to the dislocation glide (λ > 129 nm). The simulation results are highly consistent with the observed strong λ-effect, and reveal the intrinsic transition mechanisms induced by partial dislocation slip.