<p>We conducted a study to fully understand carbon dioxide (CO<sub>2</sub>) adsorption to a cylinder’s internal surface and fractionation of CO<sub>2</sub> and air during the preparation of standard mixtures of atmospheric CO<sub>2</sub> levels through a multistep dilution. The CO<sub>2</sub> molar fractions in standard mixtures prepared by diluting pure CO<sub>2</sub> with air three times deviated by −0.207 ± 0.060 μmol mol<sup>−1</sup> on average from the gravimetric values which were calculated from masses of source materials by evaluating their CO<sub>2</sub> molar fractions based on standard mixtures by diluting the pure CO<sub>2</sub> with the air only once. It indicates that the deviation is larger than a compatibility goal of 0.1 μmol mol<sup>−1</sup>, which has been recommended by the World Meteorological Organization (WMO). The deviations were consistent with those calculated from the fractionation factors of 0.99968 ± 0.00010 and 0.99975 ± 0.00004 estimated in mother–daughter transfer experiment that transfer CO<sub>2</sub>/Air mixtures from a cylinder to another evacuated receiving cylinder and by applying the Rayleigh model to the increase in CO<sub>2</sub> molar fractions in source gas as pressure depleted from 11.5 MPa to 1.1 MPa. Both fractionation factors also agree within their uncertainties. Additionally, the mother–daughter transfer experiments showed that the deviation was caused by the fractionation of CO<sub>2</sub> and air in the process of transferring a source gas (a CO<sub>2</sub>/Air mixture with a higher CO<sub>2</sub> molar fraction than that in the prepared gas mixture). The fact that the CO<sub>2</sub> molar fraction weakened significantly as the transfer speed decreased suggested that the main factor of the fractionation could be thermal diffusion. However, experiments exiting a CO<sub>2</sub> in air mixture (CO<sub>2</sub>/Air mixture) from a cylinder were conducted to evaluate the CO<sub>2</sub> adsorption to the internal surface of the cylinder. As the cylinder pressure was reduced from 11.0 to 0.1 MPa, the CO<sub>2</sub> molar fractions in the mixture flow leaving from the cylinder increased the CO<sub>2</sub> molar fractions by 0.16 ± 0.04 μmol mol<sup>−1</sup>. By applying the Langmuir adsorption-desorption model to the measured data, the amount of CO<sub>2</sub> adsorbed on the internal surfaces of a 10 L aluminum cylinder when preparing a standard mixture with atmospheric CO<sub>2</sub> level was estimated to be 0.027 ± 0.004 μmol mol<sup>−1</sup> at 11.0 MPa.</p>