Objectives: Despite numerous animal trials reporting that cell therapy promotes collateral flow, clinical trials have not convincingly shown benefit. Patient-related risk factors are often used to explain these discrepancies. However, during the course of our own angiogenesis studies using mice, we noted large anatomical variability in collateral vessels. The purpose of the present investigation was to define how important this factor might be in determining intervention outcomes. Methods: Hindlimb ischemia was induced in BalbC mice by ligating the superficial femoral artery. After 24h, animals were treated by injecting the adductor muscle with either control media or cultured mesenchymal stem cells (MSCs). Blood flow recovery was measured using laser Doppler (LDPI-ratio). In a second experiment, mice were stratified 24h after arterial ligation prior to treatment using a simple clinical score of the ligated leg: 1: able to flex, mild discoloration; 2: no flexion, mild discoloration; 3: severe discoloration; 4: any necrosis. Results: Without stratification, blood flow recovery significantly increased in the MSC treated group (p<0.05, n=6 MSC group, n=7 media group). In the experiment employing stratification, all differences between the groups disappeared (n=11 MSC group, n=10 media group; p=0.3). Furthermore, we found a striking inverse correlation between clinical score on day 1 and the LDPI-ratio on day 28 (p<0.0001; n=79). Anatomic confirmation of the disparity in preexisting collaterals was found in two different mouse strains using micro-CT. Conclusions: There is substantial inter-animal variability in preexisting collateral flow, and this variability can importantly influence outcome. To overcome this, either animals must be stratified prior to treatment, the number of animals must be increased substantially, or preferably, both.