The CreER T2 for conditional gene inactivation has become increasingly used in reverse mouse genetics, which enables temporal regulation of Cre activity using a mutant estrogen binding domain (ER T2) to keep Cre inactive until the administration of tamoxifen. In this study, we present the severe toxicity of ubiquitously expressed CreER T2 in adult mice and embryos. The toxicity of Cre recombinase or CreER T2 in vitro or in vivo organisms are still less sufficiently recognized considering the common use of Cre/loxP system, though the toxicity might compromise the phenotypic analysis of the gene of interest. We analyzed two independent lines in which CreER T2 is knocked-in into the Rosa26 locus (R26CreER T2 mice), and both lines showed thymus atrophy, severe anemia, and illegitimate chromosomal rearrangement in hematopoietic cells after the administration of tamoxifen, and demonstrated complete recovery of hematological toxicity in adult mice. In the hematopoietic tissues in R26CreER T2 mice, reduced proliferation and increased apoptosis was observed after the administration of tamoxifen. Flow cytometric analysis revealed that CreER T2 toxicity affected several hematopoietic lineages, and that immature cells in these lineages tend to be more sensitive to the toxicity. In vitro culturing of hematopoietic cells from these mice further demonstrated the direct toxicity of CreER T2 on growth and differentiation of hematopoietic cells. We further demonstrated the cleavage of the putative cryptic/pseudo loxP site in the genome after the activation of CreER T2 in vivo. We discussed how to avoid the misinterpretation of the experimental results from potential toxic effects due to the activated CreER T2.