Jak2 signaling is required for HSC production in the AGM. (A) Cryosections (10 μM) were prepared from E11.5 embryos and stained with antibodies against CD34 and total Stat5 as indicated (ventral down). Pictures were taken with a Zeiss AxioSkop2 wide-field microscope (objective 20×/045 NA) fitted with a Zeiss AxioCam MRc5, and images were analyzed with the Zeiss AxioVision software. (B) E11.5 AGM cells from embryos with the indicated genotypes were directly plated in methylcellulose, and colonies were counted 7 days later. n = 3 for Jak2^+/+; n = 4 for Jak2^+/fl; n = 3 for Jak2^fl/fl. (C) E11.5 AGM cells from embryos with the indicated genotypes were directly transplanted as 1 embryo equivalent, and donor cell contribution to the peripheral blood of the recipients was determined at 4 months. Dotted line represents 5% threshold. Fourteen recipients for Jak2^+/+; 13 recipients for Jak2^+/fl; 10 recipients for Jak2^fl/fl. **P < .01. (D) Cryosections (10 μM) were prepared from (i) E11.5 Jak2^+/+, (ii) Jak2^+/fl, and (iii) Jak2^fl/fl embryos and stained with antibodies against CD34 and Ki67, together with TUNEL staining as indicated (ventral down). n = 2 for each genotype; 16 to 21 sections were analyzed per genotype. Pictures were taken with a Zeiss Axio Imager Microscope (objective 40×) fitted with a Hammatsu Flash 4 V2 sCMOS camera, and images were analyzed with the Zen software.