Résumé : Mouse knockouts of Cdk2 and Cdk4 are individually viable whereas the double knockouts are embryonic lethal due to heart defects, and this precludes the investigation of their overlapping roles in definitive hematopoiesis. Here we use a conditional knockout mouse model to investigate the effect of combined loss of Cdk2 and Cdk4 in hematopoietic cells. Cdk2(fl/fl)Cdk4(-/-)vavCre mice are viable but displayed a significant increase in erythrocyte size. Cdk2(fl/fl)Cdk4(-/-)vavCre mouse bone marrow exhibited reduced phosphorylation of the retinoblastoma protein and reduced expression of E2F target genes such as cyclin A2 and Cdk1. Erythroblasts lacking Cdk2 and Cdk4 displayed a lengthened G1 phase due to impaired phosphorylation of the retinoblastoma protein. Deletion of the retinoblastoma protein rescued the increased size displayed by erythrocytes lacking Cdk2 and Cdk4, indicating that the retinoblastoma/Cdk2/Cdk4 pathway regulates erythrocyte size. The recovery of platelet counts following a 5-fluorouracil challenge was delayed in Cdk2(fl/fl)Cdk4(-/-)vavCre mice revealing a critical role for Cdk2 and Cdk4 in stress hematopoiesis. Our data indicate that Cdk2 and Cdk4 play important overlapping roles in homeostatic and stress hematopoiesis, which need to be considered when using broad-spectrum cyclin-dependent kinase inhibitors for cancer therapy.