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Oral cancer affects people globally with an estimated 657,000 new cases and 330,000 deaths yearly. Macrophage migration inhibitory factor (Mif) is a gene that encodes for a cytokine involved in regulating cell-mediated immunity, immunoregulation, and inflammation. Previous research has demonstrated that global Mif inhibition results in reduced oral cancer development. This was associated with a decrease in the accumulation of myeloid cell populations in the oral tumor microenvironment. It is therefore important to elucidate the role of Mif expression by myeloid populations in the oral tumor microenvironment. We previously generated floxed Mif reporter mice (Mif Fl/Fl). Selective breeding between Mif Fl/Fl mice and Cre knock-in mice (which contains a nuclear localized cre recombinase targeted to the Lysozyme 2 gene locus) (LysMCre) was performed to produce mice with Mif deficiency in myeloid cells. Genotypic characterization for Mif Fl/Fl and LysMCre was performed by PCR of genomic DNA extracted from mouse ear tissue. Phenotypic characterization of myeloid Mif deficiency was performed through flow cytometric analysis on various myeloid cell populations. Selective breeding of the Mif Fl/Fl mice with the LysMCre mice for three generations yielded myeloid-specific Mif deficient mice. Genotypic PCR characterization of Mif Fl/Fl mice yielded expected band sizes of 404 bp while wild-type mice yielded an expected band size of 221 bp. Furthermore, genotypic characterization of LysMCre mice yielded an expected band size of 700 bp while wild-type mice yielded an expected band size of 350 bp. Phenotypic flow cytometric analysis of myeloid-specific Mif deficient mice revealed deletion of Mif in bone marrow-derived macrophages and peritoneal macrophages, but not in lymphoid cells. Our data indicate that selective breeding of LysMCre mice with our recently generated Mif Fl/Fl mice results in conditional Mif gene removal in myeloid cells in mice, which can help us gain a better understanding of the role Mif plays in the context of oral cancer and other inflammatory diseases.