Thin polymer films with perpendicularly aligned nanocylinder arrays were fabricated from block copolymers of poly(N-dodecylacrylamide) and poly(ethylene glycol) (pDDA-b-PEG). Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize pDDA-b-PEG block copolymers with different degrees of polymerization (DP) of DDA by using trithiocarbonate-substituted PEG as the macrochain-transfer agent (PEG-CTA). Thin films of pDDA-b-PEG were prepared by using a spin-coating method, and their structures were characterized by atomic force microscopy (AFM) and X-ray diffraction (XRD). AFM phase images of the thermally annealed films showed a featureless surface irrespective of the DP of DDA. Subsequent annealing of the films under humid conditions induced nanophase separation of the pDDA blocks. One-dimensional XRD patterns suggested that the pDDA block formed a highly oriented lamellar structure, wherein the dodecyl side chains are perpendicularly aligned relative to the substrate. AFM images of the films annealed under humid conditions exhibited a hexagonally aligned PEG nanocylinder array. The results of two-dimensional grazing angle incident small-angle X-ray scattering (2D GI-SAXS) and cross-sectional transmission electron microscopy (TEM) image suggested a perpendicular alignment of the nanocylinders throughout the film. The diameter of the nanocylinders (similar to 8 nm) was determined by AFM and scanning transmission electron microscopy (STEM). In their entirety the results of this study suggest that the vertical orientation of the PEG nanocylinders relative to the substrate surface is induced by the orientation of the lamellar pDDA. Nanophase separation in comb-shaped polymers, in combination with a simple pDDA block and humid annealing, thus offers a unique preparative method for self-assembled structures in polymer films.