Identifying the thickness of the first graphene layer on a substrate is important in graphene-based nanoelectromechanical systems because of the dependence of graphene layers on physical and chemical properties. Identifying the thickness of the first layer is important for determining the number of graphene layers. Herein, we report that the height of single-layer graphene on mica is influenced by the relative humidity in the observation environment and by the scanning modes of in situ atomic force microscopy (AFM). We found that the graphene height is quite dependent on the scan direction of the AFM probe when in the contact mode, while this hysteresis is absent in tapping and noncontact modes. In addition, at low humidity (<10%), the height of the graphene on mica corresponds to the van der Waals distance (similar to 0.34 nm) of graphite layers, while an increased height (0.67 nm) is observed at higher humidity (>= 20%). We associate the variation of the graphene height in the contact mode with different levels of tilting of the cantilever that are clearly dependent on the scanning direction. These results suggest a standard for determining graphene thickness in humid air that is a critical factor for graphene-based electronic devices. Published under license by AIP Publishing.