The self-assembly (SA) of diblock copolymers (DBCs) based on phase separation into different morphologies of small and high-density features is widely investigated as a patterning and nanofabrication technique. The integration of conventional top-down approaches with the bottom-up SA of DBCs enables the possibility to address the gap in nanostructured lateral length standards for nanometrology, consequently supporting miniaturization processes in device fabrication. On this topic, we studied the pattern characteristic dimensions (i.e., center-to-center distance L and diameter D) of a cylinder-forming polystyrene-b-poly( methyl methacrylate) PS-b-PMMA (54 kg mol, styrene fraction 70%) DBC when confined within periodic SiO trenches of different widths (W, ranging between 75 and 600 nm) and fixed length (l, 5.7 μm). The characteristic dimensions of the PMMA cylinder structure in the confined configurations were compared with those obtained on a flat surface (L = 27.8 ± 0.5 nm, D = 13.0 ± 1.0 nm). The analysis of D as a function of W evolution indicates that the eccentricity of the PMMA cylinders decreases as a result of the deformation of the cylinder in the direction perpendicular to the trenches. The center-to-center distance in the direction parallel to the long side of the trenches (L) is equal to L measured on the flat surface, whereas the one along the short side (L) is subjected to an appreciable variation (ΔL = 5 nm) depending on W. The possibility of finely tuning L maintaining constant L paves the way to the realization of a DBC-based transfer standard for lateral length calibration with periods in the critical range between 20 and 50 nm wherein no commercial transfer standards are available. A prototype transfer standard with cylindrical holes was used to calibrate the linear correction factor c(Δx') of an atomic force microscope for a scan length of Δx' = 1 μm. The relative standard uncertainty of the correction factor was only 1.3%, and the second-order nonlinear correction was found to be significant.