Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF-/- ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF-/- colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of beta h1-globin, which continued to be expressed at a very low level. The ratio of adult beta-globin/alpha-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF-/- cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF-/- erythrocytes in adult animals must be short-lived, apparently due to the imbalance of beta-versus alpha-globin chains, leading to the precipitation of excess alpha-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF-/- ES cells of a human LCR/gamma-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.