Introduction: Blood loss resulting from vessel injury is prevented in the mammalian organism through well regulated steps in the process of hemostasis. While a fully functional primary hemostatic system can be found in healthy adults, newborns and especially preterm newborns are at an increased risk for bleeding complications including intraventricular hemorrhage. Recent studies in humans reported decreased platelet reactivity in preterm newborns, opposed to data showing shortened bleeding times in term neonates. However, up to today there is no model to examine the development of the hemostatic system and the impact of maturation on primary hemostasis. Methods: To examine platelet function and thrombus formation in yolk-sac blood vessels of developing murine fetuses, pregnant C57/Bl6 mice were anesthetized (5mg/ml ketamine, 1 mg/ml xylazine in 10 ml/kg of normal saline; intraperitoneal injection), the abdominal cavity opened, the uterus prepared and one viable fetus inside the yolk-sac exteriorized. Manual microinjection allowed administration of phototoxic FITC-dextran into fetal yolk-sac vessels, as subsequent excitation with high intensity light was applied to induce thrombus formation. Injection of fluorescent microbeads allowed determination of blood flow characteristics in fetal yolk-sac blood vessels. Time course and steps of thrombus formation were determined in fetuses of ages E13.5 to E17.5. Multiphoton-microscopy was applied to study distribution patterns of the phototoxic dye within the fetal vasculature. FACS-analysis allowed acquisition of platelet counts as well as further investigation of activation patterns of fetal platelets. Results: Rate of onset and firm vessel occluding thrombus formation in murine yolk-sac vessels was significantly lower in youngest fetuses compared to older ones (p<0,05). Additionally, reflow after primary thrombus formation appeared more frequent in youngest fetuses. Time until platelet adhesion and stable thrombus formation was prolonged in E13.5 fetuses compared to older fetuses. FACS-analysis revealed significantly decreased platelet counts in fetal blood samples compared to adult levels. Furthermore, changed platelet activation properties on thrombin stimulation could be noted in fetuses compared to adult values. Conclusion: This is the first reported in-vivo observation of platelet function and thrombus formation in the developing murine fetus using a newly established intravital microscopy model. We find that during early fetal development, decreased thrombus formation as well as reduced thrombus stability are present, while a gradual normalization of primary hemostasis can be noted towards more mature gestational ages. Therefore, our results are the first to show a developmental regulation of primary hemostasis in the living fetal mammalian organism.