To develop improved methods of transition prediction for isolated roughness based on the growth of disturbances in the roughness wake, the underlying instability mechanisms must first be understood. This paper presents a direct comparison of experimentally- observed and computed instabilities due to a cylindrical roughness in a boundary layer at Mach 6, to identify the dominant mechanism for transition. Direct numerical simulations allow a detailed analysis of the entire flow field, while experimental measurements discover the real flow physics and confirm the findings of the computations. For a large roughness height of 10.2 mm, an instability with a frequency near 21 kHz originates in the separation region upstream of the roughness. Unstable shear layers and horseshoe vortices appear to cause transition downstream of the roughness for this case. As the roughness height is reduced, there appears to be a change in the dominant instability mechanism.