Transferrin receptor (TfR) facilitates cellular iron uptake by mediating endocytosis of its ligand, iron-loaded transferrin. Although TfR is widely believed to be important for iron acquisition by all mammalian cells, direct experimental evidence is lacking. We have previously shown that mouse embryos homozygous for a disrupted transferrin receptor allele (TfR^-/-) die of anemia before embryonic day 12.5, although most other embryonic tissues appear to be developing normally. Here, we have investigated the importance of TfR postnatally, by using TfR^-/- embryonic stem cells to produce chimeric animals. We find that TfR^-/- embryonic stem cells give rise to most tissues and organs, but do not contribute to hematopoietic tissues on a wild-type C57BL/6J background, indicating that both adult erythropoiesis and lymphopoiesis require TfR. On an immunodeficient RAG2^-/- background, TfR^-/- B-cell development proceeds at least to the IgM⁺ stage, although significantly fewer IgM⁺ cells are present in peripheral lymphoid organs. Conversely, T cells lacking TfR are arrested very early in their development, at the CD4^-8^-3^- stage. These results indicate that TfR is necessary for the normal maturation of thymocytes, but that B-cell development is less severely affected by the absence of TfR. (Blood. 2003;102:3711-3718)