SummaryTransferrin, the major plasma iron carrier, mediates iron entry into cells through interaction with its receptor. Several in vitro studies have demonstrated that transferrin plays an essential role in lymphocyte division, a role attributed to its iron transport function. In the present study we used hypotransferrinaemic (Trfhpx/hpx) mice to investigate the possible involvement of transferrin in T lymphocyte differentiation in vivo. The absolute number of thymocytes was substantially reduced in Trfhpx/hpx mice, a result that could not be attributed to increased apoptosis. Moreover, the proportions of the four major thymic subpopulations were maintained and the percentage of dividing cells was not reduced. A leaky block in the differentiation of CD4− CD8− CD3− CD44− CD25+ (TN3) into CD4− CD8− CD3− CD44− CD25− (TN4) cells was observed. In addition, a similar impairment of early thymocyte differentiation was observed in mice with reduced levels of transferrin receptor. The present study demonstrates, for the first time, that transferrin itself or a pathway triggered by the interaction of transferrin with its receptor is essential for normal early T‐cell differentiation in vivo.