Zero locus of a family of morphisms of vector bundles Let $X$ be a variety and $\varphi : F_1 \to F_2$ be a morphism of vector bundles over $X$. Then it is easy to check that the locus on $X$ for which $\varphi$ vanishes is a closed subscheme of $X$. Furthermore, the ideal sheaf cutting out this locus can be described locally by trivializing both vector bundles. 
Let us try to do this in families now.
Take a family of varieties $\pi:\mathcal{X} \to B$ over some base scheme $B$. We will not assume $B$ is reduced but we can assume the usual nice things about $\pi$. Lets say $\pi$ is flat, proper and of finite presentation with nice fibers. Take a morphism of vector bundles $\varphi: F_1 \to F_2$ on $\mathcal{X}$.
We want to solve the following:

Vaguely: What is the locus in $B$ like over which $\varphi$ is precisely zero.

Observe that this is the same situation as in the very first sentence of this post, if we take $\pi = \operatorname{id}_{\mathcal{X}}$. But in our current generality we need to make things a little more precise for the question to make sense. 
Define the functor $G \subset \hom(\_,B)$ so that $T\to B$ is in $G$ iff $\varphi|_{\mathcal{X}_T}$ is the zero morphism.

Precisely: Is $G$ representable? If it is a closed subscheme, what is the ideal defining it?

 A: I am just writing my comment above as an answer.  This follows from Corollaire 7.7.8, EGA III.
Grothendieck, Alexander 
Éléments de géométrie algébrique (rédigés avec la collaboration de Jean Dieudonné) : III. Étude cohomologique des faisceaux cohérents, Seconde partie. 
Publications Mathématiques de l'IHÉS, 17 (1963), p. 5-91 
http://www.numdam.org/numdam-bin/item?id=PMIHES_1963__17__5_0 
In EGA, the result is only established if $\pi$ is projective (stronger than proper).  However, the result has been significantly generalized both in the work of Martin Olsson and the work of Max Lieblich.  By now, the projective hypothesis is unnecessary.
The point is, there exists a coherent sheaf $\mathcal{N}$ on $B$ and an equivalence between the functor $\mathcal{M}\mapsto \textit{Hom}_{\mathcal{O}_B}(\mathcal{N},\mathcal{M})$ and the functor $\mathcal{M}\mapsto\pi_*\textit{Hom}_{\mathcal{O}_{\mathcal{X}}}(F_1,F_2\otimes \pi^*\mathcal{M})$.  In particular, for $\mathcal{M}$ equal to $\mathcal{O}_B$, the section $\phi$ of $\textit{Hom}_{\mathcal{O}_{\mathcal{X}}}(F_1,F_2)$ is equivalent to a morphism $\psi:\mathcal{N}\to \mathcal{O}_B$.  The image of $\psi$ is the ideal sheaf of the closed subscheme of $B$ that represents your functor $G$.
Edit. I should add; I first learned about this particular question when I happened on it myself as a student reading through Hartshorne's book in the following variant: for a closed subscheme $C$ of $\mathcal{X}$, is the subfunctor $H$ of the Yoneda functor $h_B$ is representable by a closed subscheme, where $T\to B$ factors through $H$ if and only if $C\times_B T \to \mathcal{X}\times_B T$ is an isomorphism.  If $\phi:F_1\to \mathcal{O}_X$ is a resolution of the ideal sheaf of $C$ by a locally free sheaf, then this is a special case of your question.  I believe at the time that Brian Conrad referred me to EGA III.
The references for the generalized result of Lieblich and Olsson are as follows.  The deduction from Olsson's paper requires first translating your question into an equivalent question about the Quot functor.
MR2233719 (2008c:14022) Reviewed 
Lieblich, Max(1-PRIN) 
Remarks on the stack of coherent algebras.  
Int. Math. Res. Not. 2006, Art. ID 75273, 12 pp. 
MR2183251 (2006h:14003) Reviewed 
Olsson, Martin C.(1-IASP-SM) 
On proper coverings of Artin stacks. (English summary)  
Adv. Math. 198 (2005), no. 1, 93–106. 
