The effectiveness of radiotherapy treatments depends on the accuracy of the dose delivery process. The majority of radiotherapy courses are delivered on linear accelerators with a Multi Leaf Collimator (MLC) in 3D conformal Radiation Therapy, Intensity Modulated Radiation Therapy (IMRT) or Volumetric Modulated Arc Therapy (VMAT) modes that require accurate MLC positioning. This study investigates the MLC calibration accuracy, following manufacturer procedures for an Elekta Synergy linac with the Agility head, against the radiation focal spot offset (alignment with the collimator axis of rotation). If the radiation focal spot is not aligned ideally with the collimator axis of rotation then a systematic error can be introduced into the calibration procedure affecting absolute MLC leaf positions. Calibration of diaphrams is equally affected; however they are not investigated here. The results indicate that an estimated 0.15 mm MLC uncertainty in all MLC leaves positions can be introduced due to uncertainty of the radiation focal spot positionof 0.21 mm.