Acute promyelocytic leukemia (APL) is characterized by reciprocal balanced chromosomal translocations involving retinoic acid receptor-alpha (RARalpha). RARalpha heterodimerizes with the retinoid X receptor-alpha (RXRalpha) and transcriptionally regulates myeloid differentiation in response to ATRA (all-trans retinoic acid). Several lines of evidence suggest that APL fusion proteins interact with RXRalpha. To elucidate the role of RXRalpha in APL, we conditionally knocked out RXRalpha in the hCG-NuMA-RARalpha APL mouse model. Phenotype analysis of NuMA-RARalpha+ mice demonstrated that these mice developed a myeloproliferative disease-like myeloid leukemia within 4 months of birth. While hemizygous and homozygous RXRalpha conditional knockout mice were phenotypically normal as late as 12 months of age, we observed that the leukemic phenotype in NuMA-RARalpha+ mice was dependent on the presence of functional RXRalpha. Bone marrow promyelocyte counts were significantly reduced in NuMA-RARalpha+ mice with RXRalpha knocked down. Significant differences in the accumulations of Gr-1+ and Mac-1+ cells were also seen. We further observed that genes previously identified to be cooperating events in APL were also regulated in an RXRalpha-dependent manner. We therefore propose that the APL fusion protein NuMA-RARalpha cooperates with RXRalpha in the development of leukemia in hCG-NuMA-RARalpha transgenic mice and suggest a novel role for RXRalpha in the pathogenesis of APL.