Two austenitic stainless steels (SS) were designed to exhibit close to the largest possible difference in stacking fault energy (SFE) while staying within specifications. Neutron diffraction and scanning electron microscopy electron backscatter diffraction analysis were used to quantify stacking fault widths and deformation twinning, respectively, during room temperature straining. While the low-SFE SS exhibits much wider stacking faults (19 nm) throughout deformation as compared to the high-SFE SS (12 nm), the difference in twinning is less pronounced, with a slightly lower stress (50–100 MPa lower) and lower strain (5–10%) for twinning onset in the low-SFE SS.