This study analyzes the elastic-to-plastic transition during nanoindentation of polycrystalline iron. We conduct nanoindentation (Berkovich indenter) experiments and electron backscatter diffraction analysis to investigate the initiation of plasticity by the appearance of the pop-in phenomenon in the loading curves. Numerous load-displacement curves are statistically analyzed to identify the occurrence of pop-ins. A first pop-in can result from plasticity initiation caused by homogeneous dislocation nucleation and requires shear stresses in the range of the theoretical strength of a defect-free iron crystal. The results also show that plasticity initiation in volumes with preexisting dislocations is significantly affected by small amounts of interstitially dissolved atoms (such as carbon) that are segregated into the stress fields of dislocations, impeding their mobility. Another strong influence on the pop-in behavior is grain boundaries, which can lead to large pop-ins at relatively high indentation loads. The pop-in behavior appears to be a statistical process affected by interstitial atoms, dislocation density, grain boundaries, and surface roughness. No effect of the crystallographic orientation on the pop-in behavior can be observed.