The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms' tumor 1 suppressor gene (WT1) and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the relationship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA (siRNA) against WT1 and CML28 in leukemic cell line K562 to examine the interaction between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was detected by using RQ-PCR and Western blotting. K562 cells transfected with WT1-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by using immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide important clues for further study on the role of CML28 and WT1 in leukemic cells.