We present a combination of self-consistent field theory simulations and experimental results to explore the mechanism behind the orientational preference of second-layer cylinders in nanomeshes formed by two consecutive steps of the self-assembly of block copolymers (BCPs). Incommensurability of the top-layer cylinder spacing with that of the bottom-layer features is found to dictate orientation preference, and this mismatch can be controlled by either the film height or the nanomesh spacing ratio via the molecular weight of the polymers used. When the space available within the film does not accommodate the hexagonal packing of the parallel orientation, the system will favor orthogonal alignment of the second-layer cylinders. This behavior is robust: it is consistently observed in many experimental systems and verified here by the comparison of free energies of both states obtained from simulations. We also discuss the impact of substrate selectivity and air polymer interface selectivity on these energies and therefore their effect on the orientational selection.