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Haploid genetics has facilitated new insights into mammalian pathways and disease mechanisms. Most animal cells are diploid, and mammalian haploid cell cultures have remained elusive for a long time. Recent methodological progress has enabled the routine derivation of haploid stem cell lines from mammalian haploid embryos. Here we provide detailed protocols for the establishment, culture, and manipulation of parthenogenetic and androgenetic haploid embryonic stem cells from mouse embryos.