Mitochondria are important for normal blood-cell development, and several diseases linked to mitochondrial DNA (mtDNA) show hematological manifestations. We recently generated a mouse strain deficient in expression of the mitochondrial pyrimidine nucleoside kinase thymidine kinase 2 (Tk2), showing that these mice exhibit progressive mtDNA depletion in multiple organs. We used this mouse strain as a model for mtDNA depletion syndromes to investigate the effects of mtDNA depletion on hematopoiesis. MtDNA levels in spleen from the Tk2-deficient mice were decreased 50%, but in contrast to all other investigated organs, both thymus and peripheral blood leukocytes showed normal mtDNA levels. Analysis of peripheral blood and cell populations in spleen, thymus, and bone marrow showed normal findings in the Tk2-deficient mice. The total rates of thymidine phosphorylation-which also include phosphorylation catalyzed by cytosolic Tk 1-in both spleen and thymus from wild-type mice were >50-fold higher than in liver, brain, and muscle. In summary, our data show that blood cells are less dependent on mitochondrial Tk2 compared with several other tissues and that these cells can synthesize deoxyribonucleotides required for mtDNA replication by alternative pathways such as phosphorylation of thymidine by cytosolic Tk1.