Holocarboxylase synthetase (HLCS) catalyzes the biotinylation of five distinct biotin-dependent carboxylases and perhaps chromatin proteins. HLCS deficiency causes multiple carboxylase deficiency which results in fatal consequences unless patients are diagnosed early and treated with pharmacological doses of biotin. The objective of this study was to develop an HLCS conditional knockout (KO) mouse and assess effects of HLCS knockout on embryo survival. In the mouse, exon 8 is flanked by LoxP sites, thereby removing a catalytically important region upon recombination by Cre. HLCS conditional KO mice were backcrossed for 14 generations with C57BL/6J mice to yield Hlcstm1Jze. Fertility and weight gain were normal and no frank disease phenotypes and abnormal feeding behavior were observed in the absence of Cre. HLCS knockout was embryonic lethal when dams homozygous for both the floxed Hlcs gene and tamoxifen-inducible Cre recombinase (denoted Hlcstm1.1Jze) were injected with tamoxifen on gestational days 2.5 and 10.5. This is the first report of an HLCS conditional KO mouse, which enables studies of the roles of HLCS and biotin in intermediary metabolism.