Mitotic recombination is an effective tool for generating mutant clones in somatic tissues. Because of difficulties associated with detecting and quantifying mutant clones in mice, this technique is limited to analysis of growth-related phenotypes induced by loss function of tumor suppressor genes. Here, we used the polymorphic CD45.1/CD45.2 alleles on chromosome 1 as pan-hematopoietic markers to track mosaic clones generated through mitotic recombination in developing T cells. We show that lineage-specific mitotic recombination can be induced and reliably detected as CD45.1 or CD45.2 homozygous clones from the CD45.1/CD45.2 heterozygous background. We have applied this system in the analysis of a lethal mutation in the Dhx9 gene. Mosaic analysis revealed a stage-specific role for Dhx9 during T-cell maturation. Thus, the experimental system described in this study offers a practical means for mosaic analysis of germline mutations in the hematopoietic system.