B-Myb is an essential transcription factor involved in control of the cell cycle and the regulation of tissue-specific gene expression in a wide range of cell types. Loss of both alleles results in early embryonic lethality at E4.5-6.5. To address the function of B-Myb in later stages of embryogenesis and in specific adult tissues, a floxed B-myb allele (B-mybF) was generated. Cre-mediated deletion in vivo was demonstrated by breeding with a transgenic GATA-Cre mouse line. An intermediate allele produced in the creation of the floxed allele, in which the PGK-neo(R) cassette is present in intron 3 (B-myb(loxneo)), was deduced to be a weak hypomorph based on the later embryonic death of homozygotes compared to B-myb(-/-) embryos. To demonstrate the efficiency and possible consequences of B-myb inactivation, we performed conditional deletion in cultured MEFs and observed decreased growth that correlated with aberrant nuclear DNA replication.