A calibration sample for transmission electron microscopy (TEM) has been developed that performs the three major instrument calibrations for a transmission electron microscope: the image magnification calibration for measurements of images, the camera constant calibration for indexing diffraction patterns, and the image/diffraction pattern rotation calibration for relating crystal directions to features in the image. This offers an improvement over commercially available calibration standards, where up to five different samples are required to perform these three calibrations. The new calibration sample consists of an electron-transparent cross-sectional TEM sample made from a molecular beam epitaxy (MBE)-grown, single-crystal semiconductor wafer. When the calibration structure is viewed in a TEM, it appears as a series of light and dark layers where the layer thicknesses are very accurately known. The calibrated thickness measurements of these light (silicon) and dark (SiGe alloy) layers are based on careful TEM measurements of the  lattice spacing of silicon which is visible on the calibration sample itself, and are supported by X-ray diffraction measurements. Furthermore, the layer thickness variation across the entire silicon wafer has been verified to be less than 1%, allowing all samples prepared from the same wafer to have errors in the given layer thickness values of less than 1%. As the sample is a single crystal of silicon, the calibrations requiring electron diffraction information such as the camera constant calibration and the image/diffraction pattern rotation calibration can also be performed easily and unambiguously. One single calibration sample can therefore be used to provide all three of the major TEM instrument calibrations at all magnifications and all camera lengths.