The fibroblast growth factor (FGF) signaling family controls a broad spectrum of cellular processes in development and adult tissue homeostasis and function, which is expressed in almost all tissues at all stages. FGF receptor substrate 2 alpha (FRS2α) is an adaptor protein that recruits downstream substrates to the FGF receptor (FGFR) tyrosine kinase. Disruption of Frs2α gene in mice abrogates activation of the mitogen‐activated protein kinase pathway by the FGFR and leads to embryonic lethality at day E7.5 post copulation. To circumvent the embryonic lethality resulting from disruption of the Frs2α gene, which hinders further characterization of the role of FRS2α in adult tissue function and homeostasis, we generated an Frs2α conditional null allele for temporally‐ and tissue‐specific disruption of the Frs2α gene. Using gene targeting in mouse embryonic stem cells, we introduced two loxP sites flanking the largest coding exon, exon 5, in the Frs2α allele. Our results indicate that the floxed Frs2α (Frs2α^flox) allele is a true conditional null allele that encodes wildtype activity and is converted to a null allele after Cre recombinase mediated recombination. genesis 45:554–559, 2007. © 2007 Wiley‐Liss, Inc.