Overwintering colonies of the allodapine bee Exoneura bicolor are characterized by marked reproductive differentiation among female nestmates. Previous work has shown that reproductively dominant females are nearly always the first females to reach adult eclosion (i.e. to hatch from the pupal case) among their brood cohort. This study examines whether this relationship is causal, or whether it reflects a correlation between eclosion order and other unknown factors that may be the actual determinants of dominance. Female pupae were transferred from natural to artificial observation nests prior to adult eclosion. Eclosion order among these females was investigated in experimental colonies which were subjected to one of four treatments. In one treatment pupae were allowed to eclose without experimental manipulation. In two treatments the development of older pupae was delayed by selective refrigeration, and in the last treatment older pupae were removed entirely. There were no effects of treatment type and no interaction between treatment type and eclosion order. In all treatments, first eclosed females became reproductively dominant.