Developing mouse models of hemophilia A has been shown to facilitate in vivo studies to explore the probable mechanism(s) underlying the disease and to examine the efficiency of the relevant potential therapeutics. This study aimed to knockout (KO) the coagulation factor viii (fviii) gene in NMRI mice, using CRISPR/Cas9 (D10A/nickase) system, to generate a mouse model of hemophilia A. Two single guide RNAs (sgRNAs), designed from two distinct regions on NMRI mouse FVIII (mFVIII) exon 3, were designed and inserted in the pX335 vector, expressing both sgRNAs and nickase. The recombinant construct was delivered into mouse zygotes and implanted into the pseudopregnant female mice's uterus. Mutant mice were identified by genotyping, genomic sequencing, and mFVIII activity assessment. Two separate lines of hemophilia A were obtained through interbreeding the offspring of the female mice receiving potential CRISPR-Cas9-edited zygotes. Genomic DNA analysis revealed disruptions of the mfviii gene reading frame through a 22-bp deletion and a 23-bp insertion in two separate founder mice. The founder mice showed all the clinical signs of hemophilia A including; excessive bleeding after injuries, and spontaneous bleeding in joints and other organs. Coagulation test data showed that mFVIII coagulation activity was significantly diminished in the mFVIII knockout (FVIIIKO) mice compared to normal mice. The CRISPR/nickase system was successfully applied to generate mouse lines with the knockout fviii gene. The two novel FVIIIKO mice demonstrated all clinical symptoms of hemophilia A, which could be successfully inherited. Therefore, both of the developed FVIIIKO mouse lines are eligible for being considered as proper mouse models of hemophilia A for in vivo therapeutic studies.