CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock-out mice. This represents creation of the first gene knock-out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock-in (Clic1FN) allele, followed by Clic1 knock-out (Clic1−/−) mice by crossing Clic1FN allele with TNAP-cre mice, resulting in germline gene deletion through Cre-mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y₁₂ receptor signaling.