State-space multivariate dynamical systems (MDS) (Ryali et al. 2011) and other causal estimation models are being increasingly used to identify directed functional interactions between brain regions. However, the validity and accuracy of such methods are poorly understood. Performance evaluation based on computer simulations of small artificial causal networks can address this problem to some extent, but they often involve simplifying assumptions that reduce biological validity of the resulting data. Here, we use a novel approach taking advantage of recently developed optogenetic fMRI (ofMRI) techniques to selectively stimulate brain regions while simultaneously recording high-resolution whole-brain fMRI data. ofMRI allows for a more direct investigation of causal influences from the stimulated site to brain regions activated downstream and is therefore ideal for evaluating causal estimation methods in vivo. We used ofMRI to investigate whether MDS models for fMRI can accurately estimate causal functional interactions between brain regions. Two cohorts of ofMRI data were acquired, one at Stanford University and the University of California Los Angeles (Cohort 1) and the other at the University of North Carolina Chapel Hill (Cohort 2). In each cohort, optical stimulation was delivered to the right primary motor cortex (M1). General linear model analysis revealed prominent downstream thalamic activation in Cohort 1, and caudate-putamen (CPu) activation in Cohort 2. MDS accurately estimated causal interactions from M1 to thalamus and from M1 to CPu in Cohort 1 and Cohort 2, respectively. As predicted, no causal influences were found in the reverse direction. Additional control analyses demonstrated the specificity of causal interactions between stimulated and target sites. Our findings suggest that MDS state-space models can accurately and reliably estimate causal interactions in ofMRI data and further validate their use for estimating causal interactions in fMRI. More generally, our study demonstrates that the combined use of optogenetics and fMRI provides a powerful new tool for evaluating computational methods designed to estimate causal interactions between distributed brain regions.