Genetically manipulated transgenic and gene‐targeted mouse models are indispensible tools used in defining the role of genes in development and organ function. Similarly, cells isolated from these mice bear the same genetic alterations and therefore can be extremely useful for studying the molecular and cellular mechanisms and regulatory gene networks involving the mutated gene under well defined culture conditions. In this unit, detailed protocols regarding isolation of mouse embryonic fibroblasts (MEFs) from mouse embryos, culture of MEFs, and immortalization of MEFs are described. These protocols should be particularly useful to those investigators who intend to establish primarily cultured MEFs and to immortalize primary MEFs into stable cell cultures with a permanent growth feature from wild‐type and mutant mouse embryos.