Hemoglobinopathies are some of the most common monogenic disorders in the world, affecting millions of people and representing a growing burden on health systems worldwide. Although the pathophysiology of sickle cell anemia and beta-thalassemia, two of the most common hemoglobinopathies, have been the focus of much research over the last century, patients affected by these diseases still lack a widely applicable and easily available cure. Sickle cell anemia and beta-thalassemia are caused by defects in the structure and production of the beta-globin chains that, along with the alpha-globin chains make up the heterotetrameric hemoglobin molecule. Studies geared towards re-expression of the silenced fetal gamma-globin gene in adult erythroid cells as a therapeutic strategy to alleviate the symptoms of beta-globin deficiencies have met with some success for the treatment of sickle cell anemia but not for beta-thalassemia. A better understanding of normal gamma-globin gene regulation will undoubtedly advance the development of more effective therapeutic strategies. Because many of the potential targets that may be modulated to achieve gamma-globin re-expression also have functions in erythroid cells other than regulating the gamma-globin gene, it is imperative to understand their role in all aspects of erythropoiesis before they are used for therapy. The current study focuses on the role of two Krüppel-like transcription factors, KLF1 and KLF2, which have known roles in the processes of primitive and definitive erythropoiesis as well as globin gene regulation. The regulation of primitive erythropoiesis by KLF1 and KLF2 is studied using the mouse as a model system because it is not possible to study primitive erythropoiesis in humans. Previous studies have shown that KLF1 and KLF2 are essential for and have overlapping roles in primitive erythropoiesis. Simultaneous ablation of KLF1 and KLF2 results in a severely anemic embryonic phenotype that is not evident in KLF1 or KLF2 single knockout embryos. In this study, we show that this anemia is caused by a paucity of blood cells, and exacerbated by diminished beta-like globin gene expression. The anemia phenotype is dose-dependent, and interestingly, can be ameliorated by a single copy of the KLF2, but not the KLF1 gene. The roles of KLF1 and KLF2 in maintaining both normal peripheral blood cell numbers and globin mRNA amounts are erythroid cell-specific. It was discovered that KLF2 has an essential function in erythroid precursor maintenance. KLF1 can partially compensate for KLF2 in this role, but is uniquely crucial for erythroid precursor proliferation, through its regulation of G1- to S-phase cell cycle transition. A more drastic impairment of primitive erythroid colony formation from embryonic progenitor cells occurs with simultaneous deficiency of KLF1 and KLF2, than with loss of a single factor. The regulation of human beta-like globin gene expression is studied using a recently developed in vitro system for the production of erythroid cells from umbilical cord blood hematopoietic precursor cells, representing a more “fetal” model of globin gene expression. Previous studies have shown that KLF1 binds to the promoters of the gamma- and beta-globin genes, while KLF2 binds to the promoter of the gamma-globin gene in cord blood-derived erythroid cells. Studies using transgenic mice carrying the entire human beta-globin locus had indicated that KLF1 and KLF2 positively regulate gamma-globin expression in mouse embryonic erythroid cells. We demonstrate in this study that KLF1 appears to have dual roles in the regulation of gamma-globin expression in human cord blood-derived definitive erythroid cells. Partial depletion of KLF1 causes elevated gamma-globin expression, while nearly complete depletion of KLF1 results in a down-regulation of gamma-globin expression. Of particular interest was the observation that KLF2 positively regulates gamma-globin expression in cord blood-derived erythroid cells. Surprisingly, KLF2 also positively regulates beta-globin expression in these cells. If regulation of gamma-globin by KLF2 proves to be a direct effect, KLF2 will join a very small group of factors known to directly activate gamma-globin expression.