The James Webb Space Telescope’s (Webb’s) deployable primary and secondary mirrors are actively controlled to achieve and maintain precise optical alignment on-orbit. Each of the 18 primary mirror segment assemblies (PMSAs) and the secondary mirror assembly (SMA) are controlled in six degrees of freedom by using six linear actuators in a hexapod arrangement. In addition, each PMSA contains a seventh actuator that adjusts radius of curvature (RoC). The actuators are of a novel stepper motor-based cryogenic two-stage design that is capable of sub-10 nm motion accuracy over a 20 mm range. The nm-level motion of the 132 actuators were carefully tested and characterized before integration into the mirror assemblies. Using these test results as an initial condition, knowledge of each actuator’s length (and therefore mirror position) has relied on software bookkeeping and configuration control to keep an accurate motor step count from which actuator position can be calculated. These operations have been carefully performed through years of Webb test operations using both ground support actuator control software as well as the flight Mirror Control Software (MCS). While the actuator’s coarse stage length is cross-checked using a linear variable differential transformer (LVDT), no on-board cross-check exists for the nm-level length changes of the actuators’ fine stage. To ensure that the software bookkeeping of motor step count is still accurate after years of testing and to test that the actuator position knowledge was properly handed off from the ground software to the flight MCS, a series of optical tests were devised and performed through the Center of Curvature (CoC) ambient optical test campaigns at the Goddard Space Flight Center (GSFC) and during the thermal-vacuum tests of the entire optical payload that were conducted in Chamber A at Johnson Space Center (JSC). In each test, the actuator Fine Step Count (FSC) value is compared to an external measurement provided by an optical metrology tool with the goal of either confirming the MCS database value, or providing a recommendation for an updated calibration if the measured FSC differs significantly from the MCS-based expectation. During ambient testing of the PMSA hexapods, the nm-level actuator length changes were measured with a custom laser deflectometer by measuring tilts of the PMSA. The PMSA RoC fine stage characterization was performed at JSC using multi-wave interferometric measurements with the CoC Optical Assembly (COCOA). Finally, the SMA hexapod fine stage characterization test was performed at JSC using the NIRCam instrument in the “pass-and-a-half” test configuration using a test source from the Aft-Optics System Source Plate Assembly (ASPA). In this paper, each of these three tests, subsequent data analyses, and uncertainty estimations will be presented. Additionally, a summary of the ensemble state of Webb’s actuator fine stages is provided, along with a comparison to a Wavefront Sensing and Control (WFSC)-based requirement for FSC errors as they relate to the optical alignment convergence of the telescope on-orbit.